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 :
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:
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 :
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 µμl and 200 μl pipettes
Method:
1. Use a Globalmix with :
Volumes (μl)
dNTP
25
Mgcl2
25
Buffer 10X
50
RNAse free water
36.5
Final
50
2. For the next step use :
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: Verification of the content of the fractions obtained from the Ni-NTA column chromatography.
Protocol: follow in this link
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 :
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
Aim: Get back the DNA amplified by PCR.
Protocol: follow in this link
Aim: Increase the quantity of DNA for cloning.
Protocol: follow in this link
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 :
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#8260;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:
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 :
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: Check if our plasmid was inserted in the bacteria, and to recover larger amounts of it.
Protocol: follow in this link
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 :
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 :
A1
A2
B1
B2
Vinserts (μl)
1
1
1
1
Vglobalmix
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 μ ;l 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 :
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: Get DNA back.
Protocol: follow in this link
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 :
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 :
1
2
3
4
5
6
7
8
9
10
11
12
13
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:
1
2
3
4
5
6
7
8
9
10
11
12
13
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
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: Put the insert into the plasmid and transform the plasmid into bacteria.
Protocol: follow in this link
Results (on the August 4, 2016)
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.
Aim: Get back the DNA from bacteria.
Protocol: follow in this link
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 :
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 :
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 :
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 :
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: Have more bacteria with the insert A.
Protocol: follow in this link
What we did in the lab: Materials
• 50 ml Falcon
• LB medium
• 20 ml and 20 μl pipettes
• TOPO
• C1 v2 and C2 v2
• Carbenicillin 50 mg⁄ml
• Incubator
• LB medium
Method:
1. In a 50 ml Falcon, put 15 ml of LB medium
2. Add 15 μl of carbenicillin
3. Add a colony in the Falcon. But for C1 v2 (2) we had to wait one more night because bacteria had not grown enough
4. Let it incubate at 37°C
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 :
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 :
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 :
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:
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: Increase the DNA.
Protocol: follow in this link
What we did in the lab: Materials
• C1⁄C2
• Na Acetate (NaOAc)
• Ethanol (70%)
• Incubator
• Buffer E
• 200 μl and 1000 μl pipette
Method:
1. Calculate the final volume for each insert :
For C1 : 50×7 = 350 μl
For C2 : 50×5 = 250 μl
2. Add 1⁄10 of the volume of NaOAc and 2.5x volume of ethanol :
For C1 : 35 μl of NaOAc and 875 μl of ethanol
For C2 : 25 μl of NaOAc and 625 μl of ethanol
3. Mix the samples and incubate 8minutes at -80°C
4. Centrifuge 5 minutes at 4°C and 15000 RPM. The, throw out the supernatant
5. Add 1 ml of ethanol ice cold (70%)
6. Centrifuge 15 minutes at 4°C and 15000 RPM
7. Throw out the supernatant and let dry at room temperature
8. Resuspend the pellet in 50 μl of buffer E
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 :
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 :
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 1#956;l and 20 μl pipettes
Method:
1. In a 1.5 ml eppendorf, put :
C1
C2
pET 43.1 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.
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
Aim: Put our ligated plasmid into bacteria.
Protocol: follow in this link
Results
48 hours later, no colony had grown so the transformation did not work, it has to be redone.
