Difference between revisions of "Team:Pasteur Paris/Microbiology week9"

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click on the weeks

Week 9

August 1, 2016:

PCR of C1 v2 and C2 v2 with Takara enzyme

Electrophoresis of the PCR products (C1 v2 and C2 v2)

August 2, 2016:

PCR of A1, A2, B1, B2, D1, D2, E1 and C2

Analysis of PCR products from August 1

Ligation of PCR products with TOPO cloning

Transformation in TOP10 competent cells

August 3, 2016:

Analysis of PCR products from August 2, 2016

Miniprep of cultures C1 v2 and C2 v2 from August 2

Electrophoresis of C1 v2 and C2 v2

Ligation of C1.2 /C1.5 and C2.1/C2.2 with pET43.1 and transformation with TOP10 competent cells

August 4, 2016:

Digestion of B1⁄B2, E1⁄E2 and pET43.1 with Hind III and Xba I

Dephosphorylation of digested pET43.1

PCR of A1⁄A2 and D1⁄D2 without MgCl₂

Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2)

Electrophoresis of PCR products A1⁄A2 and D1⁄D2

Pool of inserts C1 v2 and C2 v2

August 5, 2016:

Ligation of C1 and C2 with pET43.1 digested and dephosphorylated

Transformation of TOP10 competent cells

Ligation of B1⁄B2⁄C1⁄C2 in pET43.1

Transformation of B1⁄B2⁄C1⁄C2 in TOP10

Aim: Check if the sillification is working with different volume of HCl and TEOS.

Protocol: follow in this link

What we did in the lab:
Materials:
• 1.5 ml Eppendorfs
• Silification protein
• HCl 1 M
• TEOS
• Shaking incubator
• 15 ml Falcon
• Buffer A
• 10 μl and 200 μl pipettes

Method:
1. In a 1.5 ml Eppendorf prepare a sample with our protein, add 1 ml of HCl, 65.8 μl of TEOS and let it incubate for 4 minutes in the rotating incubator 2. In a 15 ml Falcon, put 9 ml of buffer A and add the previous sample 3. Follow if silification initiates and progresses at various times :

Table 1
Times	Comments
10 minutes	Ø
15 minutes	Microscopic crystalline specs appear
30 minutes	Microscopic crystalline specs continue to appear
50 minutes	Microscopic crystalline specs continue to appears + crystalline specs grow and form a precipitate

4. Redo this experiment with 1 ml of TEOS, 50 μl of protein and vortex:

Table 2
Times	Comments
15 minutes	Crystalline specs appear
2 hours	Crystalline specs continue to appear

5. Redo the experiment with 1 ml of TEOS to see the catalytic activity of the protein :

Table 3
Times	Comments
1h45	Ø

6. Redo the experiment with 50 μl of HCl and 50 μl of protein
7.Redo the experiment with 50 μl of HCl but without our protein

Results:
When glycerol is added to the sample, as an amphiphilic modifier, an emulsion takes place which mean that glycerol act as a surfactant.
Our protein is a catalyst

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• dNTP mix
• MgCl₂ 25 mM
• EX polymerase buffer 10X
• RNAse free water
• C1⁄C2 insert DNA
• primers For and Rev
• Takara EX DNA polymerase
• PCR machine
• 10 μl and 200 μl pipettes

Method:
1. Use a Globalmix with :

Table 4
	Volumes (μl)
dNTP	25
Mgcl₂	25
Buffer 10X	50
RNAse free water	36.5
Final	50

2. For the next step use :

Table 5
Tube	Name	Premix (μl)	C1 (μl)	C2 (μl)	Primer For (μl)	Primer Rev (μl)
1	C1 insert (For +Rev)	46.5	1	Ø	1	1
2	C1 For	46.5	1	Ø	1	Ø
3	C1 Rev	46.5	1	Ø	Ø	1
4	C2 insert (For +Rev)	46.5	Ø	1	1	1
5	C2 For	46.5	Ø	1	1	Ø
6	C2 Rev	46.5	Ø	1	Ø	1
7	Without DNA	46.5	Ø	Ø	1	1
8	C1 without primer	46.5	1	Ø	Ø	Ø
9	C2 without primer	46.5	Ø	1	Ø	Ø

3. For the PCR program, start at 24°C, add 0.5 μl of Takara enzyme and proceed to 94°C

“”/ — Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Loading buffer 6X
• PCR products
• Inserts C1 v2, C2 v2
• Primer S
• Primer AS
• Electrophoresis chamber, and power supply
• 10 μl pipette

Method:
1. Add 5 μl of DNA and 1 μl of buffer 6X to each sample 2. Use 6 µl of molecular weight marker and deposit each sample in the wells :

Table 6

L1 L2 L3 L4 L5 L6 L7 L8 L10 L11 L12

M. weight Marker Ø C1 + insert C1 + primer S C1 + primer AS C2 + insert C2 + primer S C2 + primer AS Without DNA C1 without primer C2 without primer

2. Start the electrophoresis at 50 V and 0.01 A for 10 minutes, then put it at 90 V and 0.03 A

Result
Figure 1

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Agarose
• Ethidium bromide drop
• Molecular weight ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• PCR products
• Electrophoresis chamber, power supply
• 10 μl pipette

Method:
1. Make an agarose gel at 0.7% 2. Prepare the samples with 5 μl of PCR products and 1 μl of loading buffer 6X 3. Depose each samples in the wells :

Table 7
L1	L2	L3	L4	L5	L6	L7	L8	L9	L10	L11	L12	L13	L14	L15
Ladder	Ø	A1	A2	Ø	B1	B2	Ø	D1	Ø	D2	Ø	E1	E2	Ø

4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30

Result
There are streaks in lanes 3, 4, 9 and 11 which mean that the PCR did not work. But it seems to work for lanes 6, 7, 13 and 14.
The PCR must be done another time for A1⁄A2 and D11⁄D2 with a lower temperature (1°C).

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Qiagen PCR clean up kit • PCR products • 200 μl pipette

Method:
1. Prepare the 8 samples:

Table 8
Samples	A1	A2	B1	B2	D1	D2	E1	E2
Buffer A (μl) <	90	90	90	90	90	90	90	90

2. Follow the protocol of the Qiagen PCR clean up kit

Result
We obtained 25 μl of each insert purified by PCR.

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• PCR products
• Salt solution
• pET43.1 vector DNA
• Incubator 37°C
• 10 µl pipettes
• 1 ml Eppendorfs

Method:
1 For each inserts, prepare the following mix :

Table 9
	Volumes (μl)
PCR products	4
Salt solution	1
pET 43.1	1
Total	6

2. Let ligate for 5 minutes at room temperature 3. Incubate for 10 minutes at 65°C.

Aim: Increase the quality of DNA.

Protocol: follow in this link

What we did in the lab:
Materials:
• dNTP
• MgCl₂
• Buffer 20X
• RNAse free
• 10 μl , 20 µl and 200 μl pipettes
• 1.5 ml Eppendorfs
• A1⁄A2 and B1⁄B2 inserts
• PCR machine
• 10 μl 20 µl and 200 μl pipettes
• 1.5 ml Eppendorfs

Method:
1. Prepare a Globalmix with :

Table 10
	Volumes (μl)
dNTP	12.5
MgCl₂	12.5
Buffer 20X	25
RNAse free	182.5
Primer S (For)	5
Primer AS (Rev)	5
Total	242.5

2. Prepare the following samples :

Table 11
	A1	A2	B1	B2
V_inserts (μl)	1	1	1	1
V_globalmix	48.5	48.5	48.5	48.5

3. Launch the PCR

Aim: Check the efficiency of the PCR.

Protocol: follow in this link

What we did in the lab:
Materials:
• Agarose
• Ethidium bromide drops
• Molecular weight ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• PCR products
• 10 μl pipette
• Electrophoresis machine
• 1.5 ml Eppendorfs

Method:
1. Make an agarose gel at 0.7% 2. Prepare the samples with 5 μl of PCR products and 1 μl of loading buffer 6X
3. Deposit each sample in the wells :

Table 12
L1	L2	L3	L4	L5	L6	L7	L8
Ladder (6 μl)	Ø	A1 : 5 μl of DNA + 1 μl of buffer 6X	A2 : 5 μl of DNA + 1 μl of buffer 6X	Ø	D1 : 5 μl of DNA + 1 μl of buffer 6X	D2 : 5 μl of DNA + 1 μl of buffer 6X	Ø

4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30.

Aim: Make the inserts fitted with the plasmid.

Protocol: follow in this link

What we did in the lab:
Materials:
• Hind III (NEB)
• Xba I 5NEB)
• Buffer CutSmart 10X (NEB)
• H₂O
• 1.5 Eppendorfs
• C1 v2 and C2 v2
• Incubator 37°C
• 20 μl and 200 μl pipettes

Method:
1. Prepare a Globalmix :

Table 13
	Volumes (μl)
HindIII	20
XbaI	20
Buffer CutSmart 10X	50
H₂O	10
Total	100

2. Put 5 μl of the mix in each tube and add 20 μl of DNA C1 v2 and C2 v2
3. Let digest for 2 hours at 37°C and inactivate enzymes for 5 minutes at 65°C

Aim: Check if our inserts have been correctly digested by the enzyme.

Protocol: follow in this link

What we did in the lab:
Method:
Lane 1 :

Table 14
1	2	3	4	5	6	7	8	9	10	11	12
Ladder	Ø	C1.1		Ø	C1.2		Ø	C1.3		Ø	C1.4
14	15	16	17	18	19	20	21	22	23	24	25
Ø	C1.5		Ø	C1.6		Ø	C1.7		Ø	C1.8

Lane 2:

Table 15
1	2	3	4	5	6	7	8	9	10	11	12
Ladder	Ø	C2.2		Ø	C2.3		Ø	C2.4		Ø	C2.5
14	15	16	17	18	19	20	21	22	23	24	25
Ø	C2.6		Ø	C2.8		Ø	C2.9		Ø	Ø	Ø

Aim: Get back the DNA which was digested and purified.

Protocol: follow in this link

What we did in the lab:
Materials
• Qiagen Gel Extraction Kit

Method:
Use Qiagen extraction kit with a final volume of 50 μl.

Results

Table 16
Sample	Weight of gel (g)
C1.2	1.3435
C1.4	1.3564
C1.5	1.3825
C1.6	1.4318
C1.7	1.4392
C1.8	1.3564
C1.10	1.3800
C2.1	1.3179
C2.2	1.2500
C2.3	1.3910
C2.9	1.3668
C2.10	1.3934

Aim: Prepare the ligation.

Protocol: follow in this link

What we did in the lab:
Materials
• 1.5 ml Eppendorfs
• DNA
• Buffer CutSmart (NEB)
• HindIII (NEB)
• XbaI (NEB)
• H₂O
• Samples B1⁄B2, E1⁄E2
• 10 μl and 20 μl pipettes

Method:
1. Make a master mix with :

Table 17
Volumes (μl)	pET 43.1	B1⁄B2⁄E1⁄E2
DNA	7.5	7.5
Buffer CutSmart	2.5	7.5
HindIII	1	1
XbaI	1	1
H₂O	1.5	0.5
Total	25	25

2. Distribute the volumes in each tube

Aim: Avoid the religation of the plasmid with itself.

Protocol: follow in this link

What we did in the lab:
Materials
• 1.5 ml Eppendorfs
• Digested DNA
• Dephosphorylase rSAP (recombinant Shrimp alkaline phosphatase) (NEB)
• Buffer CutSmart
• H₂O
• Incubator 37°C
• 10 μl and 20 μl pipettes

Method:
1. In a 1.5 ml Eppendorf, put :

Table 18
	Volumes (μl)
Digested DNA	9
Dephosphorylase	0.2
Buffer CutSmart	2
H₂O	8.8
Total	20

2. Incubate one hour at 37°C
3. Incubate 5 minutes at 65°C

Aim: Increase the quantity of DNA.

Protocol: follow in this link

What we did in the lab:
Materials
• dNTP mix
• MgCl₂ 25 mM
• Buffer 10X
• RNAse free water
• Primer S
• Primer AS
• Samples A1⁄A2 and D1⁄D2
• 10 μl , 20 μl and 200 μl pipettes
• PCR machine
• Takara DNA polymerase enzyme
• 1.5 ml Eppendorfs

Method:
1. Prepare the mix :

Table 19
	Volumes (μl)
dNTP	12.5
MgCl₂	Ø
Buffer 10X	25
RNAse free	195
Primer S	5
Primer AS	5
Total	242.5

2. To put the sample into the PCR machine, use the table :

Table 20
Tube	Names	Mix (μl)	A1 (μl)	A2 (μl)	D1 (μl)	D2 (μl)
1	A1	48.5	1	Ø	Ø	Ø
2	A2	48.5	Ø	1	Ø	Ø
3	D1	48.5	Ø	Ø	1	Ø
4	A1	48.5	Ø	Ø	Ø	1

3. For hot start PCR, launch the PCR machine and once it is at 95°C, add 0.5 μl of Takara enzyme in each tube

Aim: Check if the PCR is efficient.

Protocol: follow in this link

What we did in the lab:
Materials
• Agarose
• A1⁄A2 and D1⁄D2
• Molecular weight marker ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• 10 μl pipette

Method:
1. Make a 0.7% agarose gel
2. Add 5 μl of DNA and 1 μl of loading buffer. But we used 10 μl of DNA for the sample 4
3. Depose the sample for the electrophorese like this :

Table 21
L1	L2	L3	L4	L5	L6	L7	L8	L19
Ladder 6 μl	Ø	(1)	Ø	(2)	Ø	(3)	Ø	(4)

Results
A lot of streaks appeared so it did not work as expected. We have to redo this experiment with the gradient mode

Aim: Increase the quantity of DNA.

Protocol: follow in this link

What we did in the lab:
Materials
• dNTP mix
• MgCl₂ 25 mM
• Buffer 10X
• RNAse free
• Primer S
• Primer AS
• A1⁄A2 and D1⁄D2
• 200 μl pipette

Method:
1. Make a Globalmix with :

Table 22

Volumes (μl)

**dNTP**
72.5

**MgCl₂**
Ø

**Buffer 10X**
145

**RNAse free**
1131

**Primer S**
29

**Primer AS**
29

**Total**
1206.5

2. Prepare the samples :

Table 23

Name Samples Globalmix (μl) DNA (μl)

A1
to (7) 48.5 1 of A1

A2
(8) to (14) 48.5 1 of A2

D1
(15) to (21) 48.5 1 of D1

D2
(22) to (28) 48.5 1 of D2

3. Deposit the samples on the gel:

Table 24

A1 A2 D1 D2

A
(1) (8) (15) (22)

B
(2) (9) (16) (23)

C
(3) (10) (17) (24)

D
(4) (11) (18) (25)

E
(5) (12) (19) (26)

F
(6) (13) (20) (27)

G
(7) (14) (21) (28)

4. Make a 0.7% agarose gel and perform an electrophoresis with 5 µl of DNA and 1 µl of loading 6X buffer for each sample

Results

Aim: Make the inserts fitted with the plasmid.

Protocol: follow in this link

What we did in the lab:
Materials
• 10 μl, 20 μl and 200 μl pipette
• Hind III (NEB)
• XbaI (NEB)
• Buffer CutSmart
• H₂O
• B1⁄B2 and C1⁄C2

Method:
1. Make a Globalmix with :

Table 25
	Volumes (μl)
HindIII	45
XbaI	45
Buffer CutSmart	112.5
H₂O	67.5
Total	270

2. Distribute this global mix in each of our 4 tubes so they will contain :

Table 26
	Volumes (μl)
DNA	20
HindIII	1
XbaI	1
Buffer CutSmart	2.5
H₂O	1.5
Total	26

3. Follow the protocol for digestion.

Aim: Prepare the plasmid for the transformation.

Protocol: follow in this link

What we did in the lab:
Materials
• C1⁄C2
• pET43.1
• T4 ligase
• Buffer 10X
• H₂O
• Incubator
• 10 μl and 20 μl pipettes

Method:
1. In a 1.5 ml eppendorf, put :

Table 27
	C1	C2	pET 43.1 (a+) alone
C1 (μl)	15	Ø	Ø
C2 (μl)	Ø	15	Ø
pET 43.1 (μl)	4	4	4
T4ligase (μl)	1	1	1
Buffer 10X (μl)	2.2	2.2	2.2
H₂O (μl)	Ø	Ø	15
Total (μl)	22.2	22.2	22.2

2. Incubate for 1 hour at room temperature 3. Incubate for 10 minutes at 65°C

Aim: Express our plasmid in bacteria.

Protocol: follow in this link

What we did in the lab:
Materials
• Qiagen Extraction Kit

Method:
Use the Qiagen gel extraction kit for a final volume of 50 μl.

Table 28
Colonies	Δm (mg)
B1 colony 1	136
B1 colony 2	170
B2 colony 4	166
B2 colony 7	175
E1 colony 1	110
E1 colony 2	143
E2 colony 1	108
E2 colony 2	128

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Aim: Ligate our inserts with the plasmid.

Protocol: follow in this link

What we did in the lab:
Method:
We use the next volumes:

	Volumes (μl)
Insert (B1 or B2 or C1 or C2)	15
pET43.1 (100 ng)	2
T4 ligase	1
Buffer 10X	2
Total	20

click on the weeks

Week 9

August 1, 2016:

Silification tests

PCR of C1 v2 and C2 v2 with Takara enzyme

Agarose gel

Electrophoresis of the PCR products (C1 v2 and C2 v2)

PCR Clean up

August 2, 2016:

PCR of A1, A2, B1, B2, D1, D2, E1 and C2

Analysis of PCR products from August 1

PCR clean up

Ligation of PCR products with TOPO cloning

Transformation in TOP10 competent cells

PCR of A1⁄A2 and D1⁄D2

August 3, 2016:

Analysis of PCR products from August 2, 2016

Miniprep of cultures C1 v2 and C2 v2 from August 2

Digestion of C1 v2 and C2 v2

Electrophoresis of C1 v2 and C2 v2

DNA extraction from the gel

Ligation of C1.2 /C1.5 and C2.1/C2.2 with pET43.1 and transformation with TOP10 competent cells

August 4, 2016:

Miniprep of B1⁄B2 and E1⁄E2

Digestion of B1⁄B2, E1⁄E2 and pET43.1 with Hind III and Xba I

Dephosphorylation of digested pET43.1

PCR of A1⁄A2 and D1⁄D2 without MgCl₂

Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2)

Electrophoresis of PCR products A1⁄A2 and D1⁄D2

Next PCR of A1⁄A2 and D1⁄D2

Pool of inserts C1 v2 and C2 v2

August 5, 2016:

Digestion of B1⁄B2 and C1⁄C2

Ligation of C1 and C2 with pET43.1 digested and dephosphorylated

Transformation of TOP10 competent cells

Ligation of B1⁄B2⁄C1⁄C2 in pET43.1

Transformation of B1⁄B2⁄C1⁄C2 in TOP10

Aim: Check if the sillification is working with different volume of HCl and TEOS.

Protocol: follow in this link

What we did in the lab:
Materials:
• 1.5 ml Eppendorfs
• Silification protein
• HCl 1 M
• TEOS
• Shaking incubator
• 15 ml Falcon
• Buffer A
• 10 μl and 200 μl pipettes

Method:
1. In a 1.5 ml Eppendorf prepare a sample with our protein, add 1 ml of HCl, 65.8 μl of TEOS and let it incubate for 4 minutes in the rotating incubator 2. In a 15 ml Falcon, put 9 ml of buffer A and add the previous sample 3. Follow if silification initiates and progresses at various times :

Table 1
Times	Comments
10 minutes	Ø
15 minutes	Microscopic crystalline specs appear
30 minutes	Microscopic crystalline specs continue to appear
50 minutes	Microscopic crystalline specs continue to appears + crystalline specs grow and form a precipitate

4. Redo this experiment with 1 ml of TEOS, 50 μl of protein and vortex:

Table 2
Times	Comments
15 minutes	Crystalline specs appear
2 hours	Crystalline specs continue to appear

5. Redo the experiment with 1 ml of TEOS to see the catalytic activity of the protein :

Table 3
Times	Comments
1h45	Ø

6. Redo the experiment with 50 μl of HCl and 50 μl of protein
7.Redo the experiment with 50 μl of HCl but without our protein

Results:
When glycerol is added to the sample, as an amphiphilic modifier, an emulsion takes place which mean that glycerol act as a surfactant.
Our protein is a catalyst

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• dNTP mix
• MgCl₂ 25 mM
• EX polymerase buffer 10X
• RNAse free water
• C1⁄C2 insert DNA
• primers For and Rev
• Takara EX DNA polymerase
• PCR machine
• 10 μl and 200 μl pipettes

Method:
1. Use a Globalmix with :

Table 4
	Volumes (μl)
dNTP	25
Mgcl₂	25
Buffer 10X	50
RNAse free water	36.5
Final	50

2. For the next step use :

Table 5
Tube	Name	Premix (μl)	C1 (μl)	C2 (μl)	Primer For (μl)	Primer Rev (μl)
1	C1 insert (For +Rev)	46.5	1	Ø	1	1
2	C1 For	46.5	1	Ø	1	Ø
3	C1 Rev	46.5	1	Ø	Ø	1
4	C2 insert (For +Rev)	46.5	Ø	1	1	1
5	C2 For	46.5	Ø	1	1	Ø
6	C2 Rev	46.5	Ø	1	Ø	1
7	Without DNA	46.5	Ø	Ø	1	1
8	C1 without primer	46.5	1	Ø	Ø	Ø
9	C2 without primer	46.5	Ø	1	Ø	Ø

3. For the PCR program, start at 24°C, add 0.5 μl of Takara enzyme and proceed to 94°C

“”/ — Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Loading buffer 6X
• PCR products
• Inserts C1 v2, C2 v2
• Primer S
• Primer AS
• Electrophoresis chamber, and power supply
• 10 μl pipette

Method:
1. Add 5 μl of DNA and 1 μl of buffer 6X to each sample 2. Use 6 µl of molecular weight marker and deposit each sample in the wells :

Table 6

L1 L2 L3 L4 L5 L6 L7 L8 L10 L11 L12

M. weight Marker Ø C1 + insert C1 + primer S C1 + primer AS C2 + insert C2 + primer S C2 + primer AS Without DNA C1 without primer C2 without primer

2. Start the electrophoresis at 50 V and 0.01 A for 10 minutes, then put it at 90 V and 0.03 A

Result
Figure 1

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Agarose
• Ethidium bromide drop
• Molecular weight ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• PCR products
• Electrophoresis chamber, power supply
• 10 μl pipette

Method:
1. Make an agarose gel at 0.7% 2. Prepare the samples with 5 μl of PCR products and 1 μl of loading buffer 6X 3. Depose each samples in the wells :

Table 7
L1	L2	L3	L4	L5	L6	L7	L8	L9	L10	L11	L12	L13	L14	L15
Ladder	Ø	A1	A2	Ø	B1	B2	Ø	D1	Ø	D2	Ø	E1	E2	Ø

4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30

Result
There are streaks in lanes 3, 4, 9 and 11 which mean that the PCR did not work. But it seems to work for lanes 6, 7, 13 and 14.
The PCR must be done another time for A1⁄A2 and D11⁄D2 with a lower temperature (1°C).

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Qiagen PCR clean up kit • PCR products • 200 μl pipette

Method:
1. Prepare the 8 samples:

Table 8
Samples	A1	A2	B1	B2	D1	D2	E1	E2
Buffer A (μl) <	90	90	90	90	90	90	90	90

2. Follow the protocol of the Qiagen PCR clean up kit

Result
We obtained 25 μl of each insert purified by PCR.

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• PCR products
• Salt solution
• pET43.1 vector DNA
• Incubator 37°C
• 10 µl pipettes
• 1 ml Eppendorfs

Method:
1 For each inserts, prepare the following mix :

Table 9
	Volumes (μl)
PCR products	4
Salt solution	1
pET 43.1	1
Total	6

2. Let ligate for 5 minutes at room temperature 3. Incubate for 10 minutes at 65°C.

Aim: Increase the quality of DNA.

Protocol: follow in this link

What we did in the lab:
Materials:
• dNTP
• MgCl₂
• Buffer 20X
• RNAse free
• 10 μl , 20 µl and 200 μl pipettes
• 1.5 ml Eppendorfs
• A1⁄A2 and B1⁄B2 inserts
• PCR machine
• 10 μl 20 µl and 200 μl pipettes
• 1.5 ml Eppendorfs

Method:
1. Prepare a Globalmix with :

Table 10
	Volumes (μl)
dNTP	12.5
MgCl₂	12.5
Buffer 20X	25
RNAse free	182.5
Primer S (For)	5
Primer AS (Rev)	5
Total	242.5

2. Prepare the following samples :

Table 11
	A1	A2	B1	B2
V_inserts (μl)	1	1	1	1
V_globalmix	48.5	48.5	48.5	48.5

3. Launch the PCR

Aim: Check the efficiency of the PCR.

Protocol: follow in this link

What we did in the lab:
Materials:
• Agarose
• Ethidium bromide drops
• Molecular weight ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• PCR products
• 10 μl pipette
• Electrophoresis machine
• 1.5 ml Eppendorfs

Method:
1. Make an agarose gel at 0.7% 2. Prepare the samples with 5 μl of PCR products and 1 μl of loading buffer 6X
3. Deposit each sample in the wells :

Table 12
L1	L2	L3	L4	L5	L6	L7	L8
Ladder (6 μl)	Ø	A1 : 5 μl of DNA + 1 μl of buffer 6X	A2 : 5 μl of DNA + 1 μl of buffer 6X	Ø	D1 : 5 μl of DNA + 1 μl of buffer 6X	D2 : 5 μl of DNA + 1 μl of buffer 6X	Ø

4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30.

Aim: Make the inserts fitted with the plasmid.

Protocol: follow in this link

What we did in the lab:
Materials:
• Hind III (NEB)
• Xba I 5NEB)
• Buffer CutSmart 10X (NEB)
• H₂O
• 1.5 Eppendorfs
• C1 v2 and C2 v2
• Incubator 37°C
• 20 μl and 200 μl pipettes

Method:
1. Prepare a Globalmix :

Table 13
	Volumes (μl)
HindIII	20
XbaI	20
Buffer CutSmart 10X	50
H₂O	10
Total	100

2. Put 5 μl of the mix in each tube and add 20 μl of DNA C1 v2 and C2 v2
3. Let digest for 2 hours at 37°C and inactivate enzymes for 5 minutes at 65°C

Aim: Check if our inserts have been correctly digested by the enzyme.

Protocol: follow in this link

What we did in the lab:
Method:
Lane 1 :

Table 14
1	2	3	4	5	6	7	8	9	10	11	12
Ladder	Ø	C1.1		Ø	C1.2		Ø	C1.3		Ø	C1.4
14	15	16	17	18	19	20	21	22	23	24	25
Ø	C1.5		Ø	C1.6		Ø	C1.7		Ø	C1.8

Lane 2:

Table 15
1	2	3	4	5	6	7	8	9	10	11	12
Ladder	Ø	C2.2		Ø	C2.3		Ø	C2.4		Ø	C2.5
14	15	16	17	18	19	20	21	22	23	24	25
Ø	C2.6		Ø	C2.8		Ø	C2.9		Ø	Ø	Ø

Aim: Get back the DNA which was digested and purified.

Protocol: follow in this link

What we did in the lab:
Materials
• Qiagen Gel Extraction Kit

Method:
Use Qiagen extraction kit with a final volume of 50 μl.

Results

Table 16
Sample	Weight of gel (g)
C1.2	1.3435
C1.4	1.3564
C1.5	1.3825
C1.6	1.4318
C1.7	1.4392
C1.8	1.3564
C1.10	1.3800
C2.1	1.3179
C2.2	1.2500
C2.3	1.3910
C2.9	1.3668
C2.10	1.3934

Aim: Prepare the ligation.

Protocol: follow in this link

What we did in the lab:
Materials
• 1.5 ml Eppendorfs
• DNA
• Buffer CutSmart (NEB)
• HindIII (NEB)
• XbaI (NEB)
• H₂O
• Samples B1⁄B2, E1⁄E2
• 10 μl and 20 μl pipettes

Method:
1. Make a master mix with :

Table 17
Volumes (μl)	pET 43.1	B1⁄B2⁄E1⁄E2
DNA	7.5	7.5
Buffer CutSmart	2.5	7.5
HindIII	1	1
XbaI	1	1
H₂O	1.5	0.5
Total	25	25

2. Distribute the volumes in each tube

Aim: Avoid the religation of the plasmid with itself.

Protocol: follow in this link

What we did in the lab:
Materials
• 1.5 ml Eppendorfs
• Digested DNA
• Dephosphorylase rSAP (recombinant Shrimp alkaline phosphatase) (NEB)
• Buffer CutSmart
• H₂O
• Incubator 37°C
• 10 μl and 20 μl pipettes

Method:
1. In a 1.5 ml Eppendorf, put :

Table 18
	Volumes (μl)
Digested DNA	9
Dephosphorylase	0.2
Buffer CutSmart	2
H₂O	8.8
Total	20

2. Incubate one hour at 37°C
3. Incubate 5 minutes at 65°C

Aim: Increase the quantity of DNA.

Protocol: follow in this link

What we did in the lab:
Materials
• dNTP mix
• MgCl₂ 25 mM
• Buffer 10X
• RNAse free water
• Primer S
• Primer AS
• Samples A1⁄A2 and D1⁄D2
• 10 μl , 20 μl and 200 μl pipettes
• PCR machine
• Takara DNA polymerase enzyme
• 1.5 ml Eppendorfs

Method:
1. Prepare the mix :

Table 19
	Volumes (μl)
dNTP	12.5
MgCl₂	Ø
Buffer 10X	25
RNAse free	195
Primer S	5
Primer AS	5
Total	242.5

2. To put the sample into the PCR machine, use the table :

Table 20
Tube	Names	Mix (μl)	A1 (μl)	A2 (μl)	D1 (μl)	D2 (μl)
1	A1	48.5	1	Ø	Ø	Ø
2	A2	48.5	Ø	1	Ø	Ø
3	D1	48.5	Ø	Ø	1	Ø
4	A1	48.5	Ø	Ø	Ø	1

3. For hot start PCR, launch the PCR machine and once it is at 95°C, add 0.5 μl of Takara enzyme in each tube

Aim: Check if the PCR is efficient.

Protocol: follow in this link

What we did in the lab:
Materials
• Agarose
• A1⁄A2 and D1⁄D2
• Molecular weight marker ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• 10 μl pipette

Method:
1. Make a 0.7% agarose gel
2. Add 5 μl of DNA and 1 μl of loading buffer. But we used 10 μl of DNA for the sample 4
3. Depose the sample for the electrophorese like this :

Table 21
L1	L2	L3	L4	L5	L6	L7	L8	L19
Ladder 6 μl	Ø	(1)	Ø	(2)	Ø	(3)	Ø	(4)

Results
A lot of streaks appeared so it did not work as expected. We have to redo this experiment with the gradient mode

Aim: Make the inserts fitted with the plasmid.

Protocol: follow in this link

What we did in the lab:
Materials
• 10 μl, 20 μl and 200 μl pipette
• Hind III (NEB)
• XbaI (NEB)
• Buffer CutSmart
• H₂O
• B1⁄B2 and C1⁄C2

Method:
1. Make a Globalmix with :

Table 25
	Volumes (μl)
HindIII	45
XbaI	45
Buffer CutSmart	112.5
H₂O	67.5
Total	270

2. Distribute this global mix in each of our 4 tubes so they will contain :

Table 26
	Volumes (μl)
DNA	20
HindIII	1
XbaI	1
Buffer CutSmart	2.5
H₂O	1.5
Total	26

3. Follow the protocol for digestion.

Aim: Prepare the plasmid for the transformation.

Protocol: follow in this link

What we did in the lab:
Materials
• C1⁄C2
• pET43.1
• T4 ligase
• Buffer 10X
• H₂O
• Incubator
• 10 μl and 20 μl pipettes

Method:
1. In a 1.5 ml eppendorf, put :

Table 27
	C1	C2	pET 43.1 (a+) alone
C1 (μl)	15	Ø	Ø
C2 (μl)	Ø	15	Ø
pET 43.1 (μl)	4	4	4
T4ligase (μl)	1	1	1
Buffer 10X (μl)	2.2	2.2	2.2
H₂O (μl)	Ø	Ø	15
Total (μl)	22.2	22.2	22.2

2. Incubate for 1 hour at room temperature 3. Incubate for 10 minutes at 65°C

Aim: Express our plasmid in bacteria.

Protocol: follow in this link

What we did in the lab:
Materials
• Qiagen Extraction Kit

Method:
Use the Qiagen gel extraction kit for a final volume of 50 μl.

Table 28
Colonies	Δm (mg)
B1 colony 1	136
B1 colony 2	170
B2 colony 4	166
B2 colony 7	175
E1 colony 1	110
E1 colony 2	143
E2 colony 1	108
E2 colony 2	128

<

Aim: Ligate our inserts with the plasmid.

Protocol: follow in this link

What we did in the lab:
Method:
We use the next volumes:

	Volumes (μl)
Insert (B1 or B2 or C1 or C2)	15
pET43.1 (100 ng)	2
T4 ligase	1
Buffer 10X	2
Total	20

@@ Line 220: / Line 220: @@
      <p><h3><B> August 1, 2016:</B></h3></p>
      <p>
-         <a href="#exp1"><h4>119. Silification tests </h4></a><br/>
+         <a href="#exp1"><h4> Silification tests </h4></a><br/>
-         <a href="#exp2"><h4>120. PCR of C1 v2 and C2 v2 with Takara enzyme </h4></a><br/>
+         <a href="#exp2"><h4> PCR of C1 v2 and C2 v2 with Takara enzyme </h4></a><br/>
-         <a href="#exp3"><h4>121. Agarose gel </h4></a><br/>
+         <a href="#exp3"><h4> Agarose gel </h4></a><br/>
-         <a href="#exp4"><h4>122. Electrophoresis of the PCR products (C1 v2 and C2 v2) </h4></a><br/>
+         <a href="#exp4"><h4> Electrophoresis of the PCR products (C1 v2 and C2 v2) </h4></a><br/>
-         <a href="#exp5"><h4>123. PCR Clean up </h4></a><br/>
+         <a href="#exp5"><h4> PCR Clean up </h4></a><br/>
      </p>
      <p><h3><B> August 2, 2016:</B></h3></p>
      <p>
-         	<a href="#exp6"><h4>124. PCR of A1, A2, B1, B2, D1, D2, E1 and C2 </h4></a><br/>
+         	<a href="#exp6"><h4> PCR of A1, A2, B1, B2, D1, D2, E1 and C2 </h4></a><br/>
-         	<a href="#exp7"><h4>125. Analysis of PCR products from August 1 </h4></a><br/>
+         	<a href="#exp7"><h4> Analysis of PCR products from August 1 </h4></a><br/>
-         	<a href="#exp8"><h4>126. PCR clean up </h4></a><br/>
+         	<a href="#exp8"><h4> PCR clean up </h4></a><br/>
-         	<a href="#exp9"><h4>127. Ligation of PCR products with TOPO cloning </h4></a><br/>
+         	<a href="#exp9"><h4> Ligation of PCR products with TOPO cloning </h4></a><br/>
-	<a href="#exp10"><h4>128. Transformation in TOP10 competent cells </h4></a><br/>
+	<a href="#exp10"><h4> Transformation in TOP10 competent cells </h4></a><br/>
-<a href="#exp11"><h4>129. PCR of A1&#8260;A2 and D1&#8260;D2  </h4></a><br/>
+<a href="#exp11"><h4> PCR of A1&#8260;A2 and D1&#8260;D2  </h4></a><br/>
 </p>
      <p><h2><B> August 3, 2016:</B></h2></p>
      <p>
-         	<a href="#exp12"><h4>130. Analysis of PCR products from August 2, 2016  </h4></a><br/>
+         	<a href="#exp12"><h4> Analysis of PCR products from August 2, 2016  </h4></a><br/>
-             	<a href="#exp13"><h4>131. Miniprep of cultures C1 v2 and C2 v2 from August 2 </h4></a><br/>
+             	<a href="#exp13"><h4> Miniprep of cultures C1 v2 and C2 v2 from August 2 </h4></a><br/>
-             	<a href="#exp14"><h4>132. Digestion of C1 v2 and C2 v2 </h4></a><br/>
+             	<a href="#exp14"><h4> Digestion of C1 v2 and C2 v2 </h4></a><br/>
-             	<a href="#exp15"><h4>133. Electrophoresis of C1 v2 and C2 v2 </h4></a><br/>
+             	<a href="#exp15"><h4> Electrophoresis of C1 v2 and C2 v2 </h4></a><br/>
-             	<a href="#exp16"><h4>134. DNA extraction from the gel </h4></a><br/>
+             	<a href="#exp16"><h4> DNA extraction from the gel </h4></a><br/>
-             	<a href="#exp17"><h4>135. Ligation of C1.2 /C1.5 and C2.1/C2.2 with pET43.1 and transformation with TOP10 competent cells </h4></a><br/>
+             	<a href="#exp17"><h4> Ligation of C1.2 /C1.5 and C2.1/C2.2 with pET43.1 and transformation with TOP10 competent cells </h4></a><br/>
 </p>
      <p><h2><B>August 4, 2016:</B></h2></p>
      <p>
-         	<a href="#exp18"><h4>136. Miniprep of B1&#8260;B2 and E1&#8260;E2 </h4></a><br/>
+         	<a href="#exp18"><h4> Miniprep of B1&#8260;B2 and E1&#8260;E2 </h4></a><br/>
-         	<a href="#exp19"><h4>137. Digestion of B1&#8260;B2, E1&#8260;E2 and pET43.1 with Hind III and Xba I </h4></a><br/>
+         	<a href="#exp19"><h4> Digestion of B1&#8260;B2, E1&#8260;E2 and pET43.1 with Hind III and Xba I </h4></a><br/>
-         	<a href="#exp20"><h4>138. Dephosphorylation of digested pET43.1 </h4></a><br/>
+         	<a href="#exp20"><h4> Dephosphorylation of digested pET43.1 </h4></a><br/>
-         	<a href="#exp21"><h4>139. PCR of A1&#8260;A2 and D1&#8260;D2 without MgCl<sub>2<sub> </h4></a><br/>
+         	<a href="#exp21"><h4> PCR of A1&#8260;A2 and D1&#8260;D2 without MgCl<sub>2<sub> </h4></a><br/>
-             	<a href="#exp22"><h4>140. Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2) </h4></a><br/>
+             	<a href="#exp22"><h4> Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2) </h4></a><br/>
-<a href="#exp23"><h4>141. Electrophoresis of PCR products A1&#8260;A2 and D1&#8260;D2 </h4></a><br/>
+<a href="#exp23"><h4> Electrophoresis of PCR products A1&#8260;A2 and D1&#8260;D2 </h4></a><br/>
-<a href="#exp24"><h4>142. Next PCR of A1&#8260;A2 and D1&#8260;D2 </h4></a><br/>
+<a href="#exp24"><h4> Next PCR of A1&#8260;A2 and D1&#8260;D2 </h4></a><br/>
-<a href="#exp25"><h4>143. Pool of inserts C1 v2 and C2 v2 </h4></a><br/>
+<a href="#exp25"><h4> Pool of inserts C1 v2 and C2 v2 </h4></a><br/>
 </p>
      <p><B><h2> August 5, 2016:</B></h2></p>
      <p>
-             	<a href="#exp26"><h4>144. Digestion of B1&#8260;B2 and C1&#8260;C2  </h4></a><br/>
+             	<a href="#exp26"><h4> Digestion of B1&#8260;B2 and C1&#8260;C2  </h4></a><br/>
-             	<a href="#exp27"><h4>145. Ligation of C1 and C2 with pET43.1 digested and dephosphorylated </h4></a><br/>
+             	<a href="#exp27"><h4> Ligation of C1 and C2 with pET43.1 digested and dephosphorylated </h4></a><br/>
-             	<a href="#exp28"><h4>146. Transformation of TOP10 competent cells </h4></a><br/>
+             	<a href="#exp28"><h4> Transformation of TOP10 competent cells </h4></a><br/>
-              <a href="#exp29"><h4>147. Ligation of B1&#8260;B2&#8260;C1&#8260;C2 in pET43.1 </h4></a><br/>
+              <a href="#exp29"><h4> Ligation of B1&#8260;B2&#8260;C1&#8260;C2 in pET43.1 </h4></a><br/>
-	<a href="#exp30"><h4>148. Transformation of B1&#8260;B2&#8260;C1&#8260;C2 in TOP10 </h4></a><br/>
+	<a href="#exp30"><h4> Transformation of B1&#8260;B2&#8260;C1&#8260;C2 in TOP10 </h4></a><br/>
 </p>
@@ Line 290: / Line 290: @@
 . Follow if silification initiates and progresses at various times : <br/>
                    <table>
+<caption align="bottom" align="center">Table 1</caption>
                      <thead>
                           <tr>
@@ Line 317: / Line 318: @@
 . Redo this experiment with 1 ml of TEOS, 50 &#956;l of protein and vortex: <br/>
                    <table>
+<caption align="bottom" align="center">Table 2</caption>
                      <thead>
                           <tr>
@@ Line 336: / Line 338: @@
 . Redo the experiment with 1 ml of TEOS to see the catalytic activity of the protein : <br/>
                    <table>
+<caption align="bottom" align="center">Table 3</caption>
                      <thead>
                           <tr>
@@ Line 381: / Line 384: @@
 &bull; Takara EX DNA polymerase <br/>
 &bull; PCR machine <br/>
-&bull; 10 µ&#956;l and 200 &#956;l pipettes <br/><br/>
+&bull; 10 &#956;l and 200 &#956;l pipettes <br/><br/>
                 <U>Method:</U><br/>
 . Use a Globalmix with : <br/>
                    <table>
+<caption align="bottom" align="center">Table 4</caption>
                      <thead>
                           <tr>
@@ Line 416: / Line 420: @@
 . For the next step use : <br/>
                    <table>
+<caption align="bottom" align="center">Table 5</caption>
                      <thead>
                           <tr>
@@ Line 557: / Line 562: @@
 . Use 6 µl of molecular weight marker and deposit each sample in the wells :<br/>
                    <table>
+<caption align="bottom" align="center">Table 6</caption>
                      <thead>
                           <tr>
@@ Line 591: / Line 597: @@
 <U>Result</U><br/>
 <img src =””; alt = “”/>
+<center> Figure 1 </center>
 <br/><br/><br/>
                 </p>
@@ Line 646: / Line 653: @@
 &bull; 10 &#956;l pipette <br/><br/>
                 <U>Method:</U><br/>
-. Make an agarose gel at 0.7%
+. Make an agarose gel at 0.7&#37;
-. Prepare the samples with 5 µl of PCR products and 1 µl of loading buffer 6X
+. Prepare the samples with 5 &#956;l of PCR products and 1 &#956;l of loading buffer 6X
 . Depose each samples in the wells : <br/>
                    <table>
+<caption align="bottom" align="center">Table 7</caption>
                      <thead>
                           <tr>
@@ Line 716: / Line 724: @@
 . Prepare the 8 samples: <br/>
                    <table>
+<caption align="bottom" align="center">Table 8</caption>
                      <thead>
                           <tr>
@@ Line 745: / Line 754: @@
 . Follow the protocol of the Qiagen PCR clean up kit <br/><br/>
 <U>Result</U><br/>
-We obtained 25 µl of each insert purified by PCR.
+We obtained 25 &#956;l of each insert purified by PCR.
 <br/><br/><br/>
                 </p>
@@ Line 773: / Line 782: @@
 For each inserts, prepare the following mix : <br/>
                    <table>
+<caption align="bottom" align="center">Table 9</caption>
                      <thead>
                           <tr>
@@ Line 846: / Line 856: @@
 . Prepare a Globalmix with : <br/>
                    <table>
+<caption align="bottom" align="center">Table 10</caption>
                      <thead>
                           <tr>
@@ Line 885: / Line 896: @@
 . Prepare the following samples :
                    <table>
+<caption align="bottom" align="center">Table 11</caption>
                      <thead>
                           <tr>
@@ Line 896: / Line 908: @@
                      <tbody>
                            <tr>
-<td align="center" ; valign = “center”> <strong><p>  V<sub>inserts</sub> (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”><strong><p>  V<sub>inserts</sub> (&#956;l) </p></strong></td>
 <td align="center" ; valign = “center”> 1 </td>
 <td align="center" ; valign = “center”> 1 </td>
@@ Line 943: / Line 955: @@
 . Deposit each sample in the wells : <br/>
                    <table>
+<caption align="bottom" align="center">Table 12</caption>
                      <thead>
                           <tr>
@@ Line 1,013: / Line 1,026: @@
 . Prepare a Globalmix : <br/>
                    <table>
+<caption align="bottom" align="center">Table 13</caption>
                      <thead>
                           <tr>
@@ Line 1,063: / Line 1,077: @@
 Lane 1 : <br/>
                    <table>
+<caption align="bottom" align="center">Table 14</caption>
                      <thead>
                           <tr>
@@ Line 1,126: / Line 1,141: @@
 Lane 2: <br/>
                    <table>
+<caption align="bottom" align="center">Table 15</caption>
                      <thead>
                           <tr>
@@ Line 1,209: / Line 1,225: @@
 <U>Results</U><br/>
 <table>
+<caption align="bottom" align="center">Table 16</caption>
     <thead>
        <tr>
@@ Line 1,217: / Line 1,234: @@
     <tbody>
   <tr>
-<td align="center" ; valign = “center”> C1.2 </td>
+<td align="center" ; valign = “center”><strong><p> C1.2 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3435 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C1.4 </td>
+<td align="center" ; valign = “center”><strong><p> C1.4 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3564 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C1.5 </td>
+<td align="center" ; valign = “center”><strong><p> C1.5 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3825 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C1.6 </td>
+<td align="center" ; valign = “center”><strong><p> C1.6 </p></strong></td>
 <td align="center" ; valign = “center”> 1.4318 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C1.7 </td>
+<td align="center" ; valign = “center”><strong><p> C1.7 </p></strong></td>
 <td align="center" ; valign = “center”> 1.4392 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C1.8 </td>
+<td align="center" ; valign = “center”><strong><p> C1.8 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3564 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C1.10 </td>
+<td align="center" ; valign = “center”><strong><p> C1.10 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3800 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C2.1 </td>
+<td align="center" ; valign = “center”><strong><p> C2.1 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3179 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C2.2 </td>
+<td align="center" ; valign = “center”><strong><p> C2.2 </p></strong></td>
 <td align="center" ; valign = “center”> 1.2500 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C2.3 </td>
+<td align="center" ; valign = “center”><strong><p> C2.3 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3910 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C2.9 </td>
+<td align="center" ; valign = “center”><strong><p> C2.9 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3668 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> C2.10 </td>
+<td align="center" ; valign = “center”><strong><p> C2.10 </p></strong></td>
 <td align="center" ; valign = “center”> 1.3934 </td>
 </tr>
@@ Line 1,325: / Line 1,342: @@
 . Make a master mix with : <br/>
 <table>
+<caption align="bottom" align="center">Table 17</caption>
     <thead>
        <tr>
@@ Line 1,394: / Line 1,412: @@
 . In a 1.5 ml Eppendorf, put : <br/>
 <table>
+<caption align="bottom" align="center">Table 18</caption>
     <thead>
        <tr>
@@ Line 1,457: / Line 1,476: @@
 . Prepare the mix : <br/>
 <table>
+<caption align="bottom" align="center">Table 19</caption>
     <thead>
        <tr>
@@ Line 1,496: / Line 1,516: @@
 . To put the sample into the PCR machine, use the table : <br/>
 <table>
+<caption align="bottom" align="center">Table 20</caption>
     <thead>
        <tr>
@@ Line 1,606: / Line 1,627: @@
 . Depose the sample for the electrophorese like this : <br/>
 <table>
+<caption align="bottom" align="center">Table 21</caption>
     <thead>
        <tr>
@@ Line 1,664: / Line 1,686: @@
 . Make a Globalmix with : <br/>
 <table>
+<caption align="bottom" align="center">Table 22</caption>
     <thead>
        <tr>
@@ Line 1,703: / Line 1,726: @@
 . Prepare the samples : <br/>
 <table>
+<caption align="bottom" align="center">Table 23</caption>
     <thead>
        <tr>
@@ Line 1,740: / Line 1,764: @@
 . Deposit the samples on the gel: <br/>
 <table>
+<caption align="bottom" align="center">Table 24</caption>
     <thead>
        <tr>
@@ Line 1,868: / Line 1,893: @@
 . Make a Globalmix with : <br/>
 <table>
+<caption align="bottom" align="center">Table 25</caption>
     <thead>
        <tr>
@@ Line 1,899: / Line 1,925: @@
 . Distribute this global mix in each of our 4 tubes so they will contain : <br/>
 <table>
+<caption align="bottom" align="center">Table 26</caption>
     <thead>
        <tr>
@@ Line 1,957: / Line 1,984: @@
 &bull; H<sub>2</sub>O <br/>
 &bull; Incubator <br/>
-&bull; 10 1&#956;l and 20 &#956;l pipettes
+&bull; 10 &#956;l and 20 &#956;l pipettes
 <br/><br/>
 <U>Method:</U><br/>
 . In a 1.5 ml eppendorf, put : <br/>
 <table>
+<caption align="bottom" align="center">Table 27</caption>
     <thead>
        <tr>
@@ Line 1,967: / Line 1,995: @@
           <th> C1 </th>
           <th> C2 </th>
-          <th> pET 43.1 alone </th>
+          <th> pET 43.1 (a+) alone </th>
        </tr>
     </thead>
@@ Line 2,035: / Line 2,063: @@
 Use the Qiagen gel extraction kit for a final volume of 50 &#956;l. <br/>
 <table>
+<caption align="bottom" align="center">Table 28</caption>
     <thead>
        <tr>
@@ Line 2,043: / Line 2,072: @@
     <tbody>
   <tr>
-<td align="center" ; valign = “center”> B1 colony 1 </td>
+<td align="center" ; valign = “center”><strong><p> B1 colony 1 </p></strong></td>
 <td align="center" ; valign = “center”> 136 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> B1 colony 2 </td>
+<td align="center" ; valign = “center”><strong><p> B1 colony 2 </p></strong></td>
 <td align="center" ; valign = “center”> 170 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> B2 colony 4 </td>
+<td align="center" ; valign = “center”><strong><p> B2 colony 4 </p></strong></td>
 <td align="center" ; valign = “center”> 166 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> B2 colony 7 </td>
+<td align="center" ; valign = “center”><strong><p> B2 colony 7 </strong></p></td>
 <td align="center" ; valign = “center”> 175 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> E1 colony 1 </td>
+<td align="center" ; valign = “center”><strong><p> E1 colony 1 </strong></p></td>
 <td align="center" ; valign = “center”> 110 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> E1 colony 2 </td>
+<td align="center" ; valign = “center”><strong><p> E1 colony 2 </strong></p></td>
 <td align="center" ; valign = “center”> 143 </td>
 </tr>
 <tr>
-<td align="center" ; valign = “center”> E2 colony 1 </td>
+<td align="center" ; valign = “center”><strong><p> E2 colony 1 </p></strong></td>
 <td align="center" ; valign = “center”> 108 </td>
 </tr>
@@ Line 2,116: / Line 2,145: @@
 <tr>
 <td align="center" ; valign = “center”><strong><p> Buffer 10X </strong></p></td>
+<td align="center" ; valign = “center”> 2 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 20 </td>
+</tr>
+</tbody>
+</table>
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp30">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Put our ligated plasmid into bacteria. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+<U>Results</U><br/>
+hours later, no colony had grown so the transformation did not work, it has to be redone.
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+</body>
+</html>
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+<body>
+  <div id="week9">
+<p><h5><B>Week 9 </B></h3></p>
+    <p><h3><B> August 1, 2016:</B></h3></p>
+    <p>
+        <a href="#exp1"><h4> Silification tests </h4></a><br/>
+        <a href="#exp2"><h4> PCR of C1 v2 and C2 v2 with Takara enzyme </h4></a><br/>
+        <a href="#exp3"><h4> Agarose gel </h4></a><br/>
+        <a href="#exp4"><h4> Electrophoresis of the PCR products (C1 v2 and C2 v2) </h4></a><br/>
+        <a href="#exp5"><h4> PCR Clean up </h4></a><br/>
+    </p>
+    <p><h3><B> August 2, 2016:</B></h3></p>
+    <p>
+        	<a href="#exp6"><h4> PCR of A1, A2, B1, B2, D1, D2, E1 and C2 </h4></a><br/>
+        	<a href="#exp7"><h4> Analysis of PCR products from August 1 </h4></a><br/>
+        	<a href="#exp8"><h4> PCR clean up </h4></a><br/>
+        	<a href="#exp9"><h4> Ligation of PCR products with TOPO cloning </h4></a><br/>
+	<a href="#exp10"><h4> Transformation in TOP10 competent cells </h4></a><br/>
+<a href="#exp11"><h4> PCR of A1&#8260;A2 and D1&#8260;D2  </h4></a><br/>
+</p>
+    <p><h2><B> August 3, 2016:</B></h2></p>
+    <p>
+        	<a href="#exp12"><h4> Analysis of PCR products from August 2, 2016  </h4></a><br/>
+            	<a href="#exp13"><h4> Miniprep of cultures C1 v2 and C2 v2 from August 2 </h4></a><br/>
+            	<a href="#exp14"><h4> Digestion of C1 v2 and C2 v2 </h4></a><br/>
+            	<a href="#exp15"><h4> Electrophoresis of C1 v2 and C2 v2 </h4></a><br/>
+            	<a href="#exp16"><h4> DNA extraction from the gel </h4></a><br/>
+            	<a href="#exp17"><h4> Ligation of C1.2 /C1.5 and C2.1/C2.2 with pET43.1 and transformation with TOP10 competent cells </h4></a><br/>
+</p>
+    <p><h2><B>August 4, 2016:</B></h2></p>
+    <p>
+        	<a href="#exp18"><h4> Miniprep of B1&#8260;B2 and E1&#8260;E2 </h4></a><br/>
+        	<a href="#exp19"><h4> Digestion of B1&#8260;B2, E1&#8260;E2 and pET43.1 with Hind III and Xba I </h4></a><br/>
+        	<a href="#exp20"><h4> Dephosphorylation of digested pET43.1 </h4></a><br/>
+        	<a href="#exp21"><h4> PCR of A1&#8260;A2 and D1&#8260;D2 without MgCl<sub>2<sub> </h4></a><br/>
+            	<a href="#exp22"><h4> Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2) </h4></a><br/>
+<a href="#exp23"><h4> Electrophoresis of PCR products A1&#8260;A2 and D1&#8260;D2 </h4></a><br/>
+<a href="#exp24"><h4> Next PCR of A1&#8260;A2 and D1&#8260;D2 </h4></a><br/>
+<a href="#exp25"><h4> Pool of inserts C1 v2 and C2 v2 </h4></a><br/>
+</p>
+    <p><B><h2> August 5, 2016:</B></h2></p>
+    <p>
+            	<a href="#exp26"><h4> Digestion of B1&#8260;B2 and C1&#8260;C2  </h4></a><br/>
+            	<a href="#exp27"><h4> Ligation of C1 and C2 with pET43.1 digested and dephosphorylated </h4></a><br/>
+            	<a href="#exp28"><h4> Transformation of TOP10 competent cells </h4></a><br/>
+             <a href="#exp29"><h4> Ligation of B1&#8260;B2&#8260;C1&#8260;C2 in pET43.1 </h4></a><br/>
+	<a href="#exp30"><h4> Transformation of B1&#8260;B2&#8260;C1&#8260;C2 in TOP10 </h4></a><br/>
+</p>
+    <div class="lightbox" id="exp1">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Check if the sillification is working with different volume of HCl and TEOS. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+                   &bull; 1.5 ml Eppendorfs <br/>
+&bull; Silification protein <br/>
+&bull; HCl 1 M <br/>
+&bull; TEOS <br/>
+&bull; Shaking incubator <br/>
+&bull; 15 ml Falcon <br/>
+&bull; Buffer A <br/>
+&bull; 10 &#956;l and 200 &#956;l pipettes <br/><br/>
+               <U>Method:</U><br/>
+. In a 1.5 ml Eppendorf prepare a sample with our protein, add 1 ml of HCl, 65.8 &#956;l of TEOS and let it incubate for 4 minutes in the rotating incubator
+. In a 15 ml Falcon, put 9 ml of buffer A and add the previous sample
+. Follow if silification initiates and progresses at various times : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 1</caption>
+                    <thead>
+                         <tr>
+                             <th>Times</th>
+                             <th> Comments </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 10 minutes </p></strong></td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 15 minutes </p></strong></td>
+                             <td align="center" ; valign = “center”> Microscopic crystalline specs appear </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 30 minutes </p></strong></td>
+                             <td align="center" ; valign = “center”> Microscopic crystalline specs continue to appear </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 50 minutes </p></strong></td>
+                             <td align="center" ; valign = “center”> Microscopic crystalline specs continue to appears + crystalline specs grow and form a precipitate </td>
+                          </tr>
+</tbody>
+                   </table><br/>
+. Redo this experiment with 1 ml of TEOS, 50 &#956;l of protein and vortex: <br/>
+                  <table>
+<caption align="bottom" align="center">Table 2</caption>
+                    <thead>
+                         <tr>
+                             <th>Times</th>
+                             <th> Comments </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 15 minutes </p></strong></td>
+                             <td align="center" ; valign = “center”> Crystalline specs appear </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 2 hours </p></strong></td>
+                             <td align="center" ; valign = “center”> Crystalline specs continue to appear </td>
+                          </tr>
+</tbody>
+                   </table><br/>
+. Redo the experiment with 1 ml of TEOS to see the catalytic activity of the protein : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 3</caption>
+                    <thead>
+                         <tr>
+                             <th>Times</th>
+                             <th> Comments </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 1h45 </p></strong></td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                          </tr>
+</tbody>
+                   </table><br/>
+. Redo the experiment with 50 &#956;l of HCl and 50 &#956;l of protein<br/>
+.Redo the experiment with 50 &#956;l of HCl but without our protein<br/><br/>
+<U>Results: </U><br/>
+When glycerol is added to the sample, as an amphiphilic modifier, an emulsion takes place which mean that glycerol act as a surfactant. <br/>
+Our protein is a catalyst
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp2">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+                   &bull; dNTP mix <br/>
+&bull; MgCl<sub>2</sub> 25 mM <br/>
+&bull; EX polymerase buffer 10X <br/>
+&bull; RNAse free water <br/>
+&bull; C1&#8260;C2 insert DNA <br/>
+&bull; primers For and Rev <br/>
+&bull; Takara EX DNA polymerase <br/>
+&bull; PCR machine <br/>
+&bull; 10 &#956;l and 200 &#956;l pipettes <br/><br/>
+               <U>Method:</U><br/>
+. Use a Globalmix with : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 4</caption>
+                    <thead>
+                         <tr>
+                             <th> </th>
+                             <th> Volumes (&#956;l) </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> dNTP </p></strong></td>
+                             <td align="center" ; valign = “center”> 25 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> Mgcl<sub>2</sub> </p></strong></td>
+                             <td align="center" ; valign = “center”> 25 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> Buffer 10X </p></strong></td>
+                             <td align="center" ; valign = “center”> 50 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> RNAse free water </p></strong></td>
+                             <td align="center" ; valign = “center”> 36.5 </td>
+                          </tr>
+<tr>
+                             <td align="center" ; valign = “center”><strong><p> Final </p></strong></td>
+                             <td align="center" ; valign = “center”> 50 </td>
+                          </tr>
+</tbody>
+                   </table><br/>
+. For the next step use : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 5</caption>
+                    <thead>
+                         <tr>
+                             <th>Tube </th>
+                             <th> Name </th>
+                             <th> Premix (&#956;l) </th>
+                             <th> C1 (&#956;l) </th>
+                             <th> C2 (&#956;l) </th>
+                             <th> Primer For (&#956;l) </th>
+                             <th> Primer Rev (&#956;l) </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 1 </p></strong></td>
+                             <td align="center" ; valign = “center”> C1 insert (For +Rev) </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 2 </p></strong></td>
+                             <td align="center" ; valign = “center”> C1 For </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 3 </p></strong></td>
+                             <td align="center" ; valign = “center”> C1 Rev </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 4 </p></strong></td>
+                             <td align="center" ; valign = “center”> C2 insert (For +Rev) </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 5 </p></strong></td>
+                             <td align="center" ; valign = “center”> C2 For </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 6 </p></strong></td>
+                             <td align="center" ; valign = “center”> C2 Rev </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 7 </p></strong></td>
+                             <td align="center" ; valign = “center”> Without DNA </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 8 </p></strong></td>
+                             <td align="center" ; valign = “center”> C1 without primer </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                          </tr>
+                          <tr>
+                             <td align="center" ; valign = “center”><strong><p> 9 </p></strong></td>
+                             <td align="center" ; valign = “center”> C2 without primer </td>
+                             <td align="center" ; valign = “center”> 46.5 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> 1 </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                             <td align="center" ; valign = “center”> &#216; </td>
+                          </tr>
+</tbody>
+                   </table><br/>
+. For the PCR program, start at 24&#176;C, add 0.5 &#956;l of Takara enzyme and proceed to 94&#176;C
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp3">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Verification of the content of the fractions obtained from the Ni-NTA column chromatography. <br/> <br/>
+               <U> Protocol:</U> follow in this link
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp4">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+&bull; Loading buffer 6X <br/>
+&bull; PCR products  <br/>
+&bull; Inserts C1 v2, C2 v2  <br/>
+&bull; Primer S  <br/>
+&bull; Primer AS  <br/>
+&bull; Electrophoresis chamber, and power supply  <br/>
+&bull; 10 &#956;l pipette <br/><br/>
+               <U>Method:</U><br/>
+. Add 5 &#956;l of DNA and 1 &#956;l of buffer 6X to each sample
+. Use 6 µl of molecular weight marker and deposit each sample in the wells :<br/>
+                  <table>
+<caption align="bottom" align="center">Table 6</caption>
+                    <thead>
+                         <tr>
+                             <th> L1 </th>
+                             <th> L2 </th>
+                             <th> L3 </th>
+                             <th> L4 </th>
+                             <th> L5 </th>
+                             <th> L6 </th>
+                             <th> L7 </th>
+                             <th> L8 </th>
+                             <th> L10 </th>
+                             <th> L11 </th>
+                             <th> L12 </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”> M. weight Marker </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> C1 + insert </td>
+<td align="center" ; valign = “center”> C1 + primer S </td>
+<td align="center" ; valign = “center”> C1 + primer AS </td>
+<td align="center" ; valign = “center”> C2 + insert </td>
+<td align="center" ; valign = “center”> C2 + primer S </td>
+<td align="center" ; valign = “center”> C2 + primer AS </td>
+<td align="center" ; valign = “center”> Without DNA </td>
+<td align="center" ; valign = “center”> C1 without primer </td>
+<td align="center" ; valign = “center”> C2 without primer </td>
+                          </tr>
+</tbody>
+                   </table><br/>
+. Start the electrophoresis at 50 V and 0.01 A for 10 minutes, then put it at 90 V and 0.03 A <br/><br/>
+<U>Result</U><br/>
+<img src =””; alt = “”/>
+<center> Figure 1 </center>
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp5">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Get back the DNA amplified by PCR. <br/> <br/>
+               <U> Protocol:</U> follow in this link
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp6">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Increase the quantity of DNA for cloning. <br/> <br/>
+               <U> Protocol:</U> follow in this link
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp7">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+&bull; Agarose  <br/>
+&bull; Ethidium bromide drop  <br/>
+&bull; Molecular weight ladder (Gene ruler 1 Kb, Thermofisher) <br/>
+&bull; Loading buffer 6X  <br/>
+&bull; PCR products  <br/>
+&bull; Electrophoresis chamber, power supply  <br/>
+&bull; 10 &#956;l pipette <br/><br/>
+               <U>Method:</U><br/>
+. Make an agarose gel at 0.7&#37;
+. Prepare the samples with 5 &#956;l of PCR products and 1 &#956;l of loading buffer 6X
+. Depose each samples in the wells : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 7</caption>
+                    <thead>
+                         <tr>
+                             <th> L1 </th>
+                             <th> L2 </th>
+                             <th> L3 </th>
+                             <th> L4 </th>
+                             <th> L5 </th>
+                             <th> L6 </th>
+                             <th> L7 </th>
+                             <th> L8 </th>
+                             <th> L9 </th>
+                             <th> L10 </th>
+                             <th> L11 </th>
+                             <th> L12 </th>
+                             <th> L13 </th>
+                             <th> L14 </th>
+                             <th> L15 </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”> Ladder </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> A1 </td>
+<td align="center" ; valign = “center”> A2 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> B1 </td>
+<td align="center" ; valign = “center”> B2 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> D1 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> D2 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> E1 </td>
+<td align="center" ; valign = “center”> E2 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+                          </tr>
+</tbody>
+                   </table><br/>
+. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30 <br/><br/>
+<U>Result</U><br/>
+There are streaks in lanes 3, 4, 9 and 11 which mean that the PCR did not work. But it seems to work for lanes 6, 7, 13 and 14. <br/>
+The PCR must be done another time for A1&#8260;A2 and D11&#8260;D2 with a lower temperature (1&#176;C).
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp8">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+&bull; Qiagen PCR clean up kit
+&bull; PCR products
+&bull; 200 &#956;l pipette <br/><br/>
+               <U>Method:</U><br/>
+. Prepare the 8 samples: <br/>
+                  <table>
+<caption align="bottom" align="center">Table 8</caption>
+                    <thead>
+                         <tr>
+                             <th> Samples </th>
+                             <th> A1 </th>
+                             <th> A2 </th>
+                             <th> B1 </th>
+                             <th> B2 </th>
+                             <th> D1 </th>
+                             <th> D2 </th>
+                             <th> E1 </th>
+                             <th> E2 </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”><strong><p>  Buffer A (&#956;l) </p></strong><</td>
+<td align="center" ; valign = “center”> 90 </td>
+<td align="center" ; valign = “center”> 90 </td>
+<td align="center" ; valign = “center”> 90 </td>
+<td align="center" ; valign = “center”> 90 </td>
+<td align="center" ; valign = “center”> 90 </td>
+<td align="center" ; valign = “center”> 90 </td>
+<td align="center" ; valign = “center”> 90 </td>
+<td align="center" ; valign = “center”> 90 </td>
+</tr>
+</tbody>
+                   </table><br/>
+. Follow the protocol of the Qiagen PCR clean up kit <br/><br/>
+<U>Result</U><br/>
+We obtained 25 &#956;l of each insert purified by PCR.
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp9">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Test the specificity of primers For and Rev to obtain more insert. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+&bull; PCR products <br/>
+&bull; Salt solution <br/>
+&bull; pET43.1 vector DNA <br/>
+&bull; Incubator 37&#176;C <br/>
+&bull; 10 µl pipettes <br/>
+&bull; 1 ml Eppendorfs <br/><br/>
+               <U>Method:</U><br/>
+For each inserts, prepare the following mix : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 9</caption>
+                    <thead>
+                         <tr>
+                             <th> </th>
+                             <th> Volumes (&#956;l) </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”><strong><p>  PCR products </p></strong></td>
+<td align="center" ; valign = “center”> 4 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  Salt solution </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  pET 43.1 </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  Total </p></strong></td>
+<td align="center" ; valign = “center”> 6 </td>
+</tr>
+</tbody>
+                   </table><br/>
+. Let ligate for 5 minutes at room temperature
+. Incubate for 10 minutes at 65&#176;C.
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp10">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Check if our plasmid was inserted in the bacteria, and to recover larger amounts of it. <br/> <br/>
+               <U> Protocol:</U> follow in this link
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp11">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Increase the quality of DNA. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+&bull; dNTP <br/>
+&bull; MgCl<sub>2</sub> <br/>
+&bull; Buffer 20X <br/>
+&bull; RNAse free <br/>
+&bull; 10 &#956;l , 20 µl and 200 &#956;l pipettes <br/>
+&bull; 1.5 ml Eppendorfs <br/>
+&bull; A1&#8260;A2 and B1&#8260;B2 inserts <br/>
+&bull; PCR machine <br/>
+&bull; 10 &#956;l 20 µl and 200 &#956;l pipettes <br/>
+&bull; 1.5 ml Eppendorfs <br/><br/>
+               <U>Method:</U><br/>
+. Prepare a Globalmix with : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 10</caption>
+                    <thead>
+                         <tr>
+                             <th> </th>
+                             <th> Volumes (&#956;l) </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”><strong><p>  dNTP </p></strong></td>
+<td align="center" ; valign = “center”> 12.5</td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  MgCl<sub>2</sub> </p></strong></td>
+<td align="center" ; valign = “center”> 12.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  Buffer 20X </p></strong></td>
+<td align="center" ; valign = “center”> 25 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  RNAse free </p></strong></td>
+<td align="center" ; valign = “center”> 182.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  Primer S (For) </p></strong></td>
+<td align="center" ; valign = “center”> 5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  Primer AS (Rev) </p></strong></td>
+<td align="center" ; valign = “center”> 5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p>  Total </p></strong></td>
+<td align="center" ; valign = “center”> 242.5 </td>
+</tr>
+</tbody>
+                   </table><br/>
+. Prepare the following samples :
+                  <table>
+<caption align="bottom" align="center">Table 11</caption>
+                    <thead>
+                         <tr>
+                             <th> </th>
+                             <th> A1 </th>
+                             <th> A2 </th>
+                             <th> B1 </th>
+                             <th> B2 </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”><strong><p>  V<sub>inserts</sub> (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> V<sub>globalmix</sub> </p></strong></td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+</tr>
+</tbody>
+                   </table><br/>
+. Launch the PCR
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp12">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Check the efficiency of the PCR. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+&bull; Agarose <br/>
+&bull; Ethidium bromide drops <br/>
+&bull; Molecular weight ladder (Gene ruler 1 Kb, Thermofisher) <br/>
+&bull; Loading buffer 6X <br/>
+&bull; PCR products <br/>
+&bull; 10 &#956;l pipette <br/>
+&bull; Electrophoresis machine <br/>
+&bull; 1.5 ml Eppendorfs <br/><br/>
+               <U>Method:</U><br/>
+. Make an agarose gel at 0.7&#37;
+. Prepare the samples with 5 &#956;l of PCR products and 1 &#956;l of loading buffer 6X <br/>
+. Deposit each sample in the wells : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 12</caption>
+                    <thead>
+                         <tr>
+                             <th> L1 </th>
+                             <th> L2 </th>
+                             <th> L3 </th>
+                             <th> L4 </th>
+                             <th> L5 </th>
+                             <th> L6 </th>
+                             <th> L7 </th>
+                             <th> L8 </th>
+                         </tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”> Ladder (6 &#956;l) </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> A1 : 5 &#956;l of DNA + 1 &#956;l of buffer 6X </td>
+<td align="center" ; valign = “center”> A2 : 5 &#956;l of DNA + 1 &#956;l of buffer 6X </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> D1 : 5 &#956;l of DNA + 1 &#956;l of buffer 6X </td>
+<td align="center" ; valign = “center”> D2 : 5 &#956;l of DNA + 1 &#956;l of buffer 6X </td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+</tbody>
+                   </table><br/>
+. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30.
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp13">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Get DNA back. <br/> <br/>
+               <U> Protocol:</U> follow in this link
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp14">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Make the inserts fitted with the plasmid. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+               <U>Materials:</U><br/>
+&bull; Hind III (NEB) <br/>
+&bull; Xba I 5NEB) <br/>
+&bull; Buffer CutSmart 10X (NEB) <br/>
+&bull; H<sub>2</sub>O <br/>
+&bull; 1.5 Eppendorfs <br/>
+&bull; C1 v2 and C2 v2 <br/>
+&bull; Incubator 37&#176;C <br/>
+&bull; 20 &#956;l and 200 &#956;l pipettes <br/><br/>
+               <U>Method:</U><br/>
+. Prepare a Globalmix : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 13</caption>
+                    <thead>
+                         <tr>
+                             <th> </th>
+                             <th> Volumes (&#956;l) </th>
+</tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”><strong><p> HindIII </p></strong></td>
+<td align="center" ; valign = “center”> 20 </td>
+</tr>
+   <tr>
+<td align="center" ; valign = “center”><strong><p>  XbaI </p></strong></td>
+<td align="center" ; valign = “center”> 20 </td>
+</tr>
+   <tr>
+<td align="center" ; valign = “center”><strong><p>  Buffer CutSmart 10X </p></strong></td>
+<td align="center" ; valign = “center”> 50 </td>
+</tr>
+   <tr>
+<td align="center" ; valign = “center”><strong><p>  H<sub>2</sub>O </p></strong></td>
+<td align="center" ; valign = “center”> 10 </td>
+</tr>
+ 		  <tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 100 </td>
+</tr>
+</tbody>
+                   </table><br/>
+. Put 5 &#956;l of the mix in each tube and add 20 &#956;l of DNA C1 v2 and C2 v2 <br/>
+. Let digest for 2 hours at 37&#176;C and inactivate enzymes for 5 minutes at 65&#176;C
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp15">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Check if our inserts have been correctly digested by the enzyme. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Method:</U><br/>
+Lane 1 : <br/>
+                  <table>
+<caption align="bottom" align="center">Table 14</caption>
+                    <thead>
+                         <tr>
+                             <th> 1 </th>
+                             <th> 2 </th>
+                             <th> 3 </th>
+                             <th> 4 </th>
+                             <th> 5 </th>
+                             <th> 6 </th>
+                             <th> 7 </th>
+                             <th> 8 </th>
+                             <th> 9 </th>
+                             <th> 10 </th>
+                             <th> 11 </th>
+                             <th> 12 </th>
+                             <th> 13 </th>
+</tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”> Ladder </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.1 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.2 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.3 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.4 </td>
+</tr>
+</tbody>
+                    <thead>
+                         <tr>
+                             <th> 14 </th>
+                             <th> 15 </th>
+                             <th> 16 </th>
+                             <th> 17 </th>
+                             <th> 18 </th>
+                             <th> 19 </th>
+                             <th> 20 </th>
+                             <th> 21 </th>
+                             <th> 22 </th>
+                             <th> 23 </th>
+                             <th> 24 </th>
+                             <th> 25 </th>
+                             <th>  </th>
+</tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.5 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.6 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.7 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C1.8 </td>
+<td align="center" ; valign = “center”>  </td>
+</tr>
+</tbody>
+                   </table><br/>
+Lane 2: <br/>
+                  <table>
+<caption align="bottom" align="center">Table 15</caption>
+                    <thead>
+                         <tr>
+                             <th> 1 </th>
+                             <th> 2 </th>
+                             <th> 3 </th>
+                             <th> 4 </th>
+                             <th> 5 </th>
+                             <th> 6 </th>
+                             <th> 7 </th>
+                             <th> 8 </th>
+                             <th> 9 </th>
+                             <th> 10 </th>
+                             <th> 11 </th>
+                             <th> 12 </th>
+                             <th> 13 </th>
+</tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”> Ladder </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C2.2 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C2.3 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C2.4 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C2.5 </td>
+</tr>
+</tbody>
+                    <thead>
+                         <tr>
+                             <th> 14 </th>
+                             <th> 15 </th>
+                             <th> 16 </th>
+                             <th> 17 </th>
+                             <th> 18 </th>
+                             <th> 19 </th>
+                             <th> 20 </th>
+                             <th> 21 </th>
+                             <th> 22 </th>
+                             <th> 23 </th>
+                             <th> 24 </th>
+                             <th> 25 </th>
+                             <th>  </th>
+</tr>
+                   </thead>
+                    <tbody>
+                          <tr>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C2.6 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C2.8 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”; colspan = 2> C2.9 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+</tbody>
+                   </table>
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp16">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Get back the DNA which was digested and purified. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; Qiagen Gel Extraction Kit <br/><br/>
+<U>Method:</U><br/>
+Use Qiagen extraction kit with a final volume of 50 &#956;l. <br/><br/>
+<U>Results</U><br/>
+<table>
+<caption align="bottom" align="center">Table 16</caption>
+   <thead>
+      <tr>
+         <th> Sample </th>
+         <th> Weight of gel (g) </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> C1.2 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3435 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C1.4 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3564 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C1.5 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3825 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C1.6 </p></strong></td>
+<td align="center" ; valign = “center”> 1.4318 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C1.7 </p></strong></td>
+<td align="center" ; valign = “center”> 1.4392 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C1.8 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3564 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C1.10 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3800 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C2.1 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3179 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C2.2 </p></strong></td>
+<td align="center" ; valign = “center”> 1.2500 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C2.3 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3910 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C2.9 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3668 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C2.10 </p></strong></td>
+<td align="center" ; valign = “center”> 1.3934 </td>
+</tr>
+</tbody>
+</table>
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp17">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Put the insert into the plasmid and transform the plasmid into bacteria. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+<U>Results (on the August 4, 2016)</U><br/>
+Several colonies have grown but they are too small so we let them grow more. But C1.2 has much more colonies so we spread it into another Petri dish. <br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp18">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Get back the DNA from bacteria. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp19">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Prepare the ligation. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; 1.5 ml Eppendorfs <br/>
+&bull; DNA <br/>
+&bull; Buffer CutSmart (NEB) <br/>
+&bull; HindIII (NEB) <br/>
+&bull; XbaI (NEB) <br/>
+&bull; H<sub>2</sub>O <br/>
+&bull; Samples B1&#8260;B2, E1&#8260;E2 <br/>
+&bull; 10 &#956;l and 20 &#956;l pipettes <br/><br/>
+<U>Method:</U><br/>
+. Make a master mix with : <br/>
+<table>
+<caption align="bottom" align="center">Table 17</caption>
+   <thead>
+      <tr>
+         <th> Volumes (&#956;l) </th>
+         <th> pET 43.1 </th>
+         <th> B1&#8260;B2&#8260;E1&#8260;E2 </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> DNA </p></strong></td>
+<td align="center" ; valign = “center”> 7.5 </td>
+<td align="center" ; valign = “center”> 7.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Buffer CutSmart </p></strong></td>
+<td align="center" ; valign = “center”> 2.5 </td>
+<td align="center" ; valign = “center”> 7.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> HindIII </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> XbaI </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> H<sub>2</sub>O </p></strong></td>
+<td align="center" ; valign = “center”> 1.5 </td>
+<td align="center" ; valign = “center”> 0.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 25 </td>
+<td align="center" ; valign = “center”> 25 </td>
+</tr>
+</tbody>
+</table><br/>
+. Distribute the volumes in each tube
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp20">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Avoid the religation of the plasmid with itself. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; 1.5 ml Eppendorfs <br/>
+&bull; Digested DNA <br/>
+&bull; Dephosphorylase rSAP (recombinant Shrimp alkaline phosphatase) (NEB) <br/>
+&bull; Buffer CutSmart <br/>
+&bull; H<sub>2</sub>O <br/>
+&bull; Incubator 37&#176;C <br/>
+&bull; 10 &#956;l and 20 &#956;l pipettes
+<br/><br/>
+<U>Method:</U><br/>
+. In a 1.5 ml Eppendorf, put : <br/>
+<table>
+<caption align="bottom" align="center">Table 18</caption>
+   <thead>
+      <tr>
+         <th>  </th>
+         <th> Volumes (&#956;l) </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> Digested DNA </p></strong></td>
+<td align="center" ; valign = “center”> 9 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Dephosphorylase</p></strong></td>
+<td align="center" ; valign = “center”> 0.2 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Buffer CutSmart </p></strong></td>
+<td align="center" ; valign = “center”> 2 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> H<sub>2</sub>O </p></strong></td>
+<td align="center" ; valign = “center”> 8.8 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 20 </td>
+</tr>
+</tbody>
+</table><br/>
+. Incubate one hour at 37&#176;C<br/>
+. Incubate 5 minutes at 65&#176;C
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp21">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Increase the quantity of DNA. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; dNTP mix <br/>
+&bull; MgCl<sub>2</sub> 25 mM <br/>
+&bull; Buffer 10X <br/>
+&bull; RNAse free water <br/>
+&bull; Primer S <br/>
+&bull; Primer AS <br/>
+&bull; Samples A1&#8260;A2 and D1&#8260;D2 <br/>
+&bull; 10 &#956;l , 20 &#956;l and 200 &#956;l pipettes <br/>
+&bull; PCR machine <br/>
+&bull; Takara DNA polymerase enzyme <br/>
+&bull; 1.5 ml Eppendorfs
+<br/><br/>
+<U>Method:</U><br/>
+. Prepare the mix : <br/>
+<table>
+<caption align="bottom" align="center">Table 19</caption>
+   <thead>
+      <tr>
+         <th>  </th>
+         <th> Volumes (&#956;l) </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> dNTP </p></strong></td>
+<td align="center" ; valign = “center”> 12.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> MgCl<sub>2</sub> </p></strong></td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Buffer 10X </p></strong></td>
+<td align="center" ; valign = “center”> 25 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> RNAse free </p></strong></td>
+<td align="center" ; valign = “center”> 195 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Primer S </p></strong></td>
+<td align="center" ; valign = “center”> 5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Primer AS </p></strong></td>
+<td align="center" ; valign = “center”> 5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 242.5 </td>
+</tr>
+</tbody>
+</table><br/>
+. To put the sample into the PCR machine, use the table : <br/>
+<table>
+<caption align="bottom" align="center">Table 20</caption>
+   <thead>
+      <tr>
+         <th> Tube </th>
+         <th> Names </th>
+         <th> Mix (&#956;l) </th>
+         <th> A1 (&#956;l)  </th>
+         <th> A2 (&#956;l)  </th>
+         <th> D1 (&#956;l)  </th>
+         <th> D2 (&#956;l)  </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> 1 </p></strong></td>
+<td align="center" ; valign = “center”> A1 </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> 2 </p></strong></td>
+<td align="center" ; valign = “center”> A2 </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> &#216;</td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> 3 </p></strong></td>
+<td align="center" ; valign = “center”> D1 </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> 4 </p></strong></td>
+<td align="center" ; valign = “center”> A1 </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+</tbody>
+</table><br/>
+. For hot start PCR, launch the PCR machine and once it is at 95°C, add 0.5 &#956;l of Takara enzyme in each tube
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp22">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Have more bacteria with the insert A. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; 50 ml Falcon <br/>
+&bull; LB medium <br/>
+&bull; 20 ml and 20 &#956;l pipettes <br/>
+&bull; TOPO <br/>
+&bull; C1 v2 and C2 v2 <br/>
+&bull; Carbenicillin 50 mg&#8260;ml <br/>
+&bull; Incubator <br/>
+&bull; LB medium
+<br/><br/>
+<U>Method:</U><br/>
+. In a 50 ml Falcon, put 15 ml of LB medium <br/>
+. Add 15 &#956;l of carbenicillin <br/>
+. Add a colony in the Falcon. But for C1 v2 (2) we had to wait one more night because bacteria had not grown enough <br/>
+. Let it incubate at 37&#176;C
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp23">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Check if the PCR is efficient. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; Agarose <br/>
+&bull; A1&#8260;A2 and D1&#8260;D2 <br/>
+&bull; Molecular weight marker ladder (Gene ruler 1 Kb, Thermofisher) <br/>
+&bull; Loading buffer 6X <br/>
+&bull; 10 &#956;l pipette
+<br/><br/>
+<U>Method:</U><br/>
+. Make a 0.7&#37; agarose gel <br/>
+. Add 5 &#956;l of DNA and 1 &#956;l of loading buffer. But we used 10 &#956;l of DNA for the sample 4 <br/>
+. Depose the sample for the electrophorese like this : <br/>
+<table>
+<caption align="bottom" align="center">Table 21</caption>
+   <thead>
+      <tr>
+         <th> L1 </th>
+         <th> L2 </th>
+         <th> L3 </th>
+         <th> L4 </th>
+         <th> L5 </th>
+         <th> L6 </th>
+         <th> L7 </th>
+         <th> L8 </th>
+         <th> L19</th>
+</tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”> Ladder 6 &#956;l </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> (1) </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> (2) </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> (3) </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> (4) </td>
+</tr>
+</tbody>
+</table><br/><br/>
+<U>Results</U><br/>
+A lot of streaks appeared so it did not work as expected. We have to redo this experiment with the gradient mode
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp24">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Increase the quantity of DNA. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; dNTP mix <br/>
+&bull; MgCl<sub>2</sub> 25 mM <br/>
+&bull; Buffer 10X <br/>
+&bull; RNAse free <br/>
+&bull; Primer S <br/>
+&bull; Primer AS <br/>
+&bull; A1&#8260;A2 and D1&#8260;D2 <br/>
+&bull; 200 &#956;l pipette
+<br/><br/>
+<U>Method:</U><br/>
+. Make a Globalmix with : <br/>
+<table>
+<caption align="bottom" align="center">Table 22</caption>
+   <thead>
+      <tr>
+         <th>  </th>
+         <th> Volumes (&#956;l) </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> dNTP </p></strong></td>
+<td align="center" ; valign = “center”> 72.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> MgCl<sub>2</sub> </p></strong></td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Buffer 10X </p></strong></td>
+<td align="center" ; valign = “center”> 145 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> RNAse free </p></strong></td>
+<td align="center" ; valign = “center”> 1131 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Primer S </p></strong></td>
+<td align="center" ; valign = “center”> 29 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Primer AS </p></strong></td>
+<td align="center" ; valign = “center”> 29 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 1206.5 </td>
+</tr>
+</tbody>
+</table><br/>
+. Prepare the samples : <br/>
+<table>
+<caption align="bottom" align="center">Table 23</caption>
+   <thead>
+      <tr>
+         <th> Name </th>
+         <th> Samples </th>
+         <th> Globalmix (&#956;l)  </th>
+         <th> DNA (&#956;l)  </th>
+</tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> A1 </p></strong></td>
+<td align="center" ; valign = “center”> to (7) </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 1 of A1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> A2 </p></strong></td>
+<td align="center" ; valign = “center”> (8) to (14) </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 1 of A2 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> D1 </p></strong></td>
+<td align="center" ; valign = “center”> (15) to (21) </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 1 of D1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> D2 </p></strong></td>
+<td align="center" ; valign = “center”> (22) to (28) </td>
+<td align="center" ; valign = “center”> 48.5 </td>
+<td align="center" ; valign = “center”> 1 of D2 </td>
+</tr>
+</tbody>
+</table><br/>
+. Deposit the samples on the gel: <br/>
+<table>
+<caption align="bottom" align="center">Table 24</caption>
+   <thead>
+      <tr>
+         <th>  </th>
+         <th> A1 </th>
+         <th> A2  </th>
+         <th> D1</th>
+<th> D2</th>
+</tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> A </p></strong></td>
+<td align="center" ; valign = “center”> (1) </td>
+<td align="center" ; valign = “center”> (8) </td>
+<td align="center" ; valign = “center”> (15) </td>
+<td align="center" ; valign = “center”> (22) </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> B </p></strong></td>
+<td align="center" ; valign = “center”> (2) </td>
+<td align="center" ; valign = “center”> (9) </td>
+<td align="center" ; valign = “center”> (16) </td>
+<td align="center" ; valign = “center”> (23) </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C </p></strong></td>
+<td align="center" ; valign = “center”> (3) </td>
+<td align="center" ; valign = “center”> (10) </td>
+<td align="center" ; valign = “center”> (17) </td>
+<td align="center" ; valign = “center”> (24) </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> D </p></strong></td>
+<td align="center" ; valign = “center”> (4) </td>
+<td align="center" ; valign = “center”> (11) </td>
+<td align="center" ; valign = “center”> (18) </td>
+<td align="center" ; valign = “center”> (25) </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> E </p></strong></td>
+<td align="center" ; valign = “center”> (5) </td>
+<td align="center" ; valign = “center”> (12) </td>
+<td align="center" ; valign = “center”> (19) </td>
+<td align="center" ; valign = “center”> (26) </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> F </p></strong></td>
+<td align="center" ; valign = “center”> (6) </td>
+<td align="center" ; valign = “center”> (13) </td>
+<td align="center" ; valign = “center”> (20) </td>
+<td align="center" ; valign = “center”> (27) </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> G </p></strong></td>
+<td align="center" ; valign = “center”> (7) </td>
+<td align="center" ; valign = “center”> (14) </td>
+<td align="center" ; valign = “center”> (21) </td>
+<td align="center" ; valign = “center”> (28) </td>
+</tr>
+</tbody>
+</table><br/>
+. Make a 0.7% agarose gel and perform an electrophoresis with 5 µl of DNA and 1 µl of loading 6X buffer for each sample <br/><br/>
+<U>Results</U><br/>
+<img src = « « ; alt « « />
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp25">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Increase the DNA. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; C1&#8260;C2 <br/>
+&bull; Na Acetate (NaOAc) <br/>
+&bull; Ethanol (70&#37;) <br/>
+&bull; Incubator <br/>
+&bull; Buffer E <br/>
+&bull; 200 &#956;l and 1000 &#956;l pipette <br/>
+<br/><br/>
+<U>Method:</U><br/>
+. Calculate the final volume for each insert : <br/>
+&emsp;For C1 :	50&#215;7 = 350 &#956;l<br/>
+&emsp;For C2 :	50&#215;5 = 250 &#956;l<br/>
+. Add 1&#8260;10 of the volume of NaOAc and 2.5x volume of ethanol : <br/>
+&emsp;For C1 :	35 &#956;l of NaOAc and 875 &#956;l of ethanol <br/>
+&emsp;For C2 :	25 &#956;l of NaOAc and 625 &#956;l of ethanol <br/>
+. Mix the samples and incubate 8minutes at -80&#176;C <br/>
+. Centrifuge 5 minutes at 4&#176;C and 15000 RPM. The, throw out the supernatant <br/>
+. Add 1 ml of ethanol ice cold (70&#37;) <br/>
+. Centrifuge 15 minutes at 4&#176;C and 15000 RPM <br/>
+. Throw out the supernatant and let dry at room temperature <br/>
+. Resuspend the pellet in 50 &#956;l of buffer E
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp26">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Make the inserts fitted with the plasmid. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; 10 &#956;l, 20 &#956;l and 200 &#956;l pipette <br/>
+&bull; Hind III (NEB) <br/>
+&bull; XbaI (NEB) <br/>
+&bull; Buffer CutSmart <br/>
+&bull; H<sub>2</sub>O <br/>
+&bull; B1&#8260;B2 and C1&#8260;C2
+<br/><br/>
+<U>Method:</U><br/>
+. Make a Globalmix with : <br/>
+<table>
+<caption align="bottom" align="center">Table 25</caption>
+   <thead>
+      <tr>
+         <th>  </th>
+         <th> Volumes (&#956;l) </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> HindIII </p></strong></td>
+<td align="center" ; valign = “center”> 45 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> XbaI </p></strong></td>
+<td align="center" ; valign = “center”> 45 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Buffer CutSmart </p></strong></td>
+<td align="center" ; valign = “center”> 112.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> H<sub>2</sub>O </p></strong></td>
+<td align="center" ; valign = “center”> 67.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 270 </td>
+</tr>
+</tbody>
+</table><br/>
+. Distribute this global mix in each of our 4 tubes so they will contain : <br/>
+<table>
+<caption align="bottom" align="center">Table 26</caption>
+   <thead>
+      <tr>
+         <th> </th>
+         <th> Volumes (&#956;l)  </th>
+</tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> DNA </p></strong></td>
+<td align="center" ; valign = “center”> 20 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> HindIII </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> XbaI </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Buffer CutSmart </p></strong></td>
+<td align="center" ; valign = “center”> 2.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> H<sub>2</sub>O </p></strong></td>
+<td align="center" ; valign = “center”> 1.5 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Total </p></strong></td>
+<td align="center" ; valign = “center”> 26 </td>
+</tr>
+</tbody>
+</table><br/>
+. Follow the protocol for digestion.
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp27">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Prepare the plasmid for the transformation. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; C1&#8260;C2 <br/>
+&bull; pET43.1 <br/>
+&bull; T4 ligase <br/>
+&bull; Buffer 10X <br/>
+&bull; H<sub>2</sub>O <br/>
+&bull; Incubator <br/>
+&bull; 10 &#956;l and 20 &#956;l pipettes
+<br/><br/>
+<U>Method:</U><br/>
+. In a 1.5 ml eppendorf, put : <br/>
+<table>
+<caption align="bottom" align="center">Table 27</caption>
+   <thead>
+      <tr>
+         <th>  </th>
+         <th> C1 </th>
+         <th> C2 </th>
+         <th> pET 43.1 (a+) alone </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> C1 (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> 15 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> C2 (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> &#216;  </td>
+<td align="center" ; valign = “center”> 15 </td>
+<td align="center" ; valign = “center”> &#216; </td>
+</tr>
+<td align="center" ; valign = “center”><strong><p> pET 43.1 (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> 4 </td>
+<td align="center" ; valign = “center”> 4 </td>
+<td align="center" ; valign = “center”> 4 </td>
+</tr>
+<td align="center" ; valign = “center”><strong><p> T4ligase (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> 1 </td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<td align="center" ; valign = “center”><strong><p> Buffer 10X (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> 2.2 </td>
+<td align="center" ; valign = “center”> 2.2 </td>
+<td align="center" ; valign = “center”> 2.2 </td>
+</tr>
+<td align="center" ; valign = “center”><strong><p> H<sub>2</sub>O (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> &#216;  </td>
+<td align="center" ; valign = “center”> &#216; </td>
+<td align="center" ; valign = “center”> 15 </td>
+</tr>
+<td align="center" ; valign = “center”><strong><p> Total (&#956;l) </p></strong></td>
+<td align="center" ; valign = “center”> 22.2 </td>
+<td align="center" ; valign = “center”> 22.2 </td>
+<td align="center" ; valign = “center”> 22.2 </td>
+</tr>
+</tbody>
+</table><br/>
+. Incubate for 1 hour at room temperature
+. Incubate for 10 minutes at 65&#176;C
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp28">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Express our plasmid in bacteria. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Materials</U><br/>
+&bull; Qiagen Extraction Kit
+<br/><br/>
+<U>Method:</U><br/>
+Use the Qiagen gel extraction kit for a final volume of 50 &#956;l. <br/>
+<table>
+<caption align="bottom" align="center">Table 28</caption>
+   <thead>
+      <tr>
+         <th> Colonies </th>
+         <th> &#916;m (mg) </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strong><p> B1 colony 1 </p></strong></td>
+<td align="center" ; valign = “center”> 136 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> B1 colony 2 </p></strong></td>
+<td align="center" ; valign = “center”> 170 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> B2 colony 4 </p></strong></td>
+<td align="center" ; valign = “center”> 166 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> B2 colony 7 </strong></p></td>
+<td align="center" ; valign = “center”> 175 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> E1 colony 1 </strong></p></td>
+<td align="center" ; valign = “center”> 110 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> E1 colony 2 </strong></p></td>
+<td align="center" ; valign = “center”> 143 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> E2 colony 1 </p></strong></td>
+<td align="center" ; valign = “center”> 108 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”> E2 colony 2 </td>
+<td align="center" ; valign = “center”> 128 </td>
+</tr>
+</tbody>
+</table><
+<br/><br/><br/>
+               </p>
+            </figcaption>
+       </figure>
+    </div>
+    <div class="lightbox" id="exp29">
+       <figure>
+          <a href="#" class="closemsg"></a>
+            <figcaption>
+               <p>
+               <U> Aim:</U> Ligate our inserts with the plasmid. <br/> <br/>
+               <U> Protocol:</U> follow in this link<br/><br/>
+               <U>What we did in the lab:</U><br/>
+<U>Method:</U><br/>
+We use the next volumes: <br/>
+<table>
+   <thead>
+      <tr>
+         <th>  </th>
+         <th> Volumes (&#956;l) </th>
+      </tr>
+   </thead>
+   <tbody>
+ <tr>
+<td align="center" ; valign = “center”><strng><p> Insert (B1 or B2 or C1 or C2) </p></strong></td>
+<td align="center" ; valign = “center”> 15 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> pET43.1 (100 ng) </p></strong></td>
+<td align="center" ; valign = “center”> 2 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> T4 ligase </p></strong></td>
+<td align="center" ; valign = “center”> 1 </td>
+</tr>
+<tr>
+<td align="center" ; valign = “center”><strong><p> Buffer 10X </p></strong></td>
 <td align="center" ; valign = “center”> 2 </td>
 </tr>

	Volumes (μl)
dNTP	72.5
MgCl₂	Ø
Buffer 10X	145
RNAse free	1131
Primer S	29
Primer AS	29
Total	1206.5

Name	Samples	Globalmix (μl)	DNA (μl)
A1	to (7)	48.5	1 of A1
A2	(8) to (14)	48.5	1 of A2
D1	(15) to (21)	48.5	1 of D1
D2	(22) to (28)	48.5	1 of D2

	A1	A2	D1	D2
A	(1)	(8)	(15)	(22)
B	(2)	(9)	(16)	(23)
C	(3)	(10)	(17)	(24)
D	(4)	(11)	(18)	(25)
E	(5)	(12)	(19)	(26)
F	(6)	(13)	(20)	(27)
G	(7)	(14)	(21)	(28)

Difference between revisions of "Team:Pasteur Paris/Microbiology week9"

Revision as of 03:57, 17 October 2016

click on the weeks

Week 9

August 1, 2016:

PCR of C1 v2 and C2 v2 with Takara enzyme

Agarose gel

Electrophoresis of the PCR products (C1 v2 and C2 v2)

PCR Clean up

August 2, 2016:

Analysis of PCR products from August 1

PCR clean up

Ligation of PCR products with TOPO cloning

Transformation in TOP10 competent cells

PCR of A1⁄A2 and D1⁄D2

August 3, 2016:

Miniprep of cultures C1 v2 and C2 v2 from August 2

Digestion of C1 v2 and C2 v2

Electrophoresis of C1 v2 and C2 v2

DNA extraction from the gel

Ligation of C1.2 /C1.5 and C2.1/C2.2 with pET43.1 and transformation with TOP10 competent cells

August 4, 2016:

Digestion of B1⁄B2, E1⁄E2 and pET43.1 with Hind III and Xba I

Dephosphorylation of digested pET43.1

PCR of A1⁄A2 and D1⁄D2 without MgCl2

Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2)

Electrophoresis of PCR products A1⁄A2 and D1⁄D2

Next PCR of A1⁄A2 and D1⁄D2

Pool of inserts C1 v2 and C2 v2

August 5, 2016:

Ligation of C1 and C2 with pET43.1 digested and dephosphorylated

Transformation of TOP10 competent cells

Ligation of B1⁄B2⁄C1⁄C2 in pET43.1

Transformation of B1⁄B2⁄C1⁄C2 in TOP10

click on the weeks

Week 9

August 1, 2016:

PCR of C1 v2 and C2 v2 with Takara enzyme

Agarose gel

Electrophoresis of the PCR products (C1 v2 and C2 v2)

PCR Clean up

August 2, 2016:

Analysis of PCR products from August 1

PCR clean up

Ligation of PCR products with TOPO cloning

Transformation in TOP10 competent cells

PCR of A1⁄A2 and D1⁄D2

August 3, 2016:

Miniprep of cultures C1 v2 and C2 v2 from August 2

Digestion of C1 v2 and C2 v2

Electrophoresis of C1 v2 and C2 v2

DNA extraction from the gel

Ligation of C1.2 /C1.5 and C2.1/C2.2 with pET43.1 and transformation with TOP10 competent cells

August 4, 2016:

Digestion of B1⁄B2, E1⁄E2 and pET43.1 with Hind III and Xba I

Dephosphorylation of digested pET43.1

PCR of A1⁄A2 and D1⁄D2 without MgCl2

Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2)

Electrophoresis of PCR products A1⁄A2 and D1⁄D2

Next PCR of A1⁄A2 and D1⁄D2

Pool of inserts C1 v2 and C2 v2

August 5, 2016:

Ligation of C1 and C2 with pET43.1 digested and dephosphorylated

Transformation of TOP10 competent cells

Ligation of B1⁄B2⁄C1⁄C2 in pET43.1

Transformation of B1⁄B2⁄C1⁄C2 in TOP10

PCR of A1⁄A2 and D1⁄D2 without MgCl₂

PCR of A1⁄A2 and D1⁄D2 without MgCl₂