Team:Pasteur Paris/Microbiology week8

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Week 8

July 25, 2016:

92. Next step for the growth curve

93. Measurements of insert quantity with Nanodrop

94. Ligation of the new version of the inserts

95. PCR of C1 and C2 (v2)

July 26, 2016:

96. Midiprep of the culture made on the 25^th of July

97. Extraction and resuspension of culture made on the 25^th of July

98. Extraction gel bands C1 and C2

99. Set up the TOPO cloning ratios, ligation of C2 and C1 (v2)

July 27, 2016:

100. STAB culture

101. Electrophoresis of PCR amplifications with the two inserts

102. PCR of the new versions of C1 and C2 at 63 °C

103. Electrophoresis of PCR products from C1 and C2

104. PCR of the new versions of C1 and C2

105. PCR purification

106. TOPO10 transformation with the TOPO cloning ligation product

107. Dosage of C1 v2 and C2 v2 after PCR and purification

July 28, 2016:

108. Send the recombinant vector C2.1 (from the Midiprep of the 26^th of July) for sequencing to Eurofins-MWG

109. Observation of the transformation of C1 v2 and C2 v2 and culture of one colony

110. Preparation of Petri dishes with Xgal

111. Dosage of C1 after PCR product purification

112. Fast Protein Liquid Chromatography (FPLC)

July 29, 2016:

113. SDS PAGE gel of purified protein fractions from FPLC

114. SDS PAGE gel of purified protein fractions from FPLC

115. PCR of C1 v2 and C2 v2

116. Agarose gel Electrophoresis

117. DNA extraction with TOPO cloning colony obtained on the 27^th of July

118. DNA digestion with Xba I and Hind III

July 30, 2016:

119. Protein cryoconservation

Aim: Based on the results of silification tests, make a larger quantity of fusion protein.

Protocol: follow in this link

What we did in the lab:
Materials:

50 ml Falcon tube

Shaking incubator (INFORS HT)

Swing bucket centrifuge (JOUAN GR41)

Cultures of C1.2

carbenicillin at 50 mg/ml

LB medium

Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

; Ultrospec 3100

Method:

1.Put each of the C2 1.2 cultures in a 50 ml Falcon and centrifuge 8 minutes at 3500 RPM

2. Wash the pellet with 20 ml of LB after throwing away the supernatant

3. Centrifuge again 8 minutes at 3500 RPM and throw away the supernatant

4. Resuspend the pellet in 20 ml of LB

5. Pool the two Falcons

6. Put in the rotating incubator 4 Erlenmeyers of 2 L containing 1 L of LB, at 37°C and 150 RPM

7. Add in each Erlenmeyer 10 ml of C2 1.2 culture and 1 ml of carbenicillin at 50 mg/ml

8. Measure the OD_{600 nm} at several times :

Table 1
Times	OD_600nm
0	0.014
0 h 30	0.018
1 h 00	0.020
1 h 30	0.035
2 h 00	0.067
3 h 00	0.360
3 h 15	0.451
3 h 25	0.515
3 h 40	0.701

9. Pelleted the last measured sample for 3 minutes at 7000g. Then throw away the supernatant and store the pellet at &8722; 20 °C

10. Add 0.6 ml of iPTG in each Erlenmeyer and incubate overnight

Aim: DNA concentration measurement in order to find the correct ratios for ligation.

What we did in the lab:
Materials:
• Spectrophotometer
• Quartz cuvettes
• carbenicillin at 50 mg/ml
• LB medium
• B1 v2, B2 v2, C1 v2, C2 v2 DNA inserts
• Shaking incubator (INFORS HT)
• Microbiology equipment (37 °C incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
All the measurements are done twice to be more accurate
The preculture of vector is prepared with 3 ml of LB, 3 µl of carbenicillin and one colony of C2 1.2

Results

Table 2
DNA	pET 43.1 dephosphorylated		B1 v2		B2 v2		C1 v2		C2 v2
A₂₆₀	0.265	0.254	0.180	0.188	0.166	0.163	0.181	0.156	0.197	Ø
A₂₈₀	2.39	0.211	0.077	0.063	0.060	0.052	0.077	0.070	0.071	Ø
A_{260 ⁄ 280}	1.11	1.20	2.35	2.97	2.76	3.16	2.35	3.04	2.79	Ø
A_260⁄230	0.31	0.34	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
[ DNA] (ng ⁄ μl)	13.3	12.7	9	9.4	8.3	8.2	9	7.8	9.8	Ø

Aim: Prepare the ligation before doing the transformation with the Top10 competent cell .

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 1 ml Eppendorfs
• B1 v2 , B2 v2 , C1 v2 , C2 v2 DNA inserts
• Distilled water
• Shaking incubator (INFORS HT)
• T4 ligase
• T4 buffer

Method:
1. Prepare the following samples in a 1 ml Eppendorf, finishing with the ligase :

Table 3
	B1 v2 pET 43.1(a+) from 05 ⁄ 07	B2 v2 pET 43.1(a+) from 05 ⁄ 07	C1 v2 pET 43.1(a+) from 11 ⁄ 07	C2 v2 pET 43.1(a+) from 08 ⁄ 07
100 ng dephorphorylated ( μl)	6.7	6.7	7.7	7.7
Insert (16ng) (μl)	1.6	2	2	1.6
T4 ligase (μl)	1	1	1	1
T4 buffer (μl)	1.5	1.5	1.5	1.5
H₂O (μl)	4.2	3.8	1.8	3.2
V_final (μl)	15	15	15	15

2. Let ligate 1 hour at room temperature
3.Incubate 10 minutes at 65°C to inactivate the enzymes
4.Store all the samples at −80°C until ready to transform

Aim: Increase the amount of DNA.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipement
• Mastermix 2X
• Primer S (sense direction, Forward Primer, see sequence in PCR protocol section)
• Primers AS (anti-sense, Reverse Primer, see sequence in PCR protocol section)
• Phusion DNA polymerase
• DNA template
• Distilled water
• PCR machine

Method:
1. For each insert, make 3 tubes of 0.2 ml with thin walls and prepare the following mastermix :

Table 4
Volume (μl)	Insert	Primer S	Primer AS
Mastermix 2X	75	75	75
Primer S	3	3	Ø
Primer AS	3	Ø	3
H₂O	65.4	68.4	68.4
Phusion	0.6	0.6	0.6

2. Distribute the previous mastermix in the tubes and add the template DNA to have :

Table 5
Volume (μl)	Insert	Primer S	Primer AS	Without DNA
Mastermix	25	25	25	25
Primer S	1	1	Ø	1
Primer AS	1	Ø	1	1
Template DNA diluted 1⁄10	1	1	1	Ø
H₂O	21.8	22.8	22.8	22.8
Phusion	0.2	0.2	0.2	0.2
Total	50	50	50	50

3. Launch the PCR for 24 cycles

Aim: Get back plasmid DNA with the twinned insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• QIAGEN plasmid Midi Kit
• 2 ml Eppendorfs
• Spectrophotometer
• Ethanol 70%
• Cultures of BL21DE3 from the 25⁄07

Method:
1. When isopropanol is added, spread the volume in 5 x 2 (2 ml Eppendorfs) and centrifuge 10 minutes at 13000g
2. Add 1 ml of ethanol in each pellet and centrifuge 10 minutes at 13000g

Results:
Make 3 measurements of the concentration (1⁄200 dilution)

Table 6
	Measurement 1	Measurement 2	Measurement 3
OD₂₆₀	0.046	0.44	0.073
[DNA] (μg⁄ml)	460	4.40	730

Aim: Prepare our bacteria before the protein purification.

What we did in the lab:
Materials:
• Microbiological equipment
• PMSF at 100 mM
• Cultures of BL21DE3
• Buffer A
• 50 ml Falcons

Method:
1.Centrifuge 4x 1l of cultures 20 minutes at 4500g
2.Throw away the supernatant
3.Add 40 μl of PMSF in 40 ml of buffer A and add 5 ml of this solution in each pellet
4.Scratch the pellet to resuspend it
5.Add again 5 ml of the buffer A solution and mix the slurry until it becomes smooth, and to recover all the pellet
6.Put each product in a clean 50 ml Falcon : store at −80°C.

Table 7
Tubes	1	2	3	4
Weight (g)	15	15	15.3	16.1

Aim: Extract the bands C1 and C2 amplified with 900 bp inserts, and separate non-specific bands (700 bp).

Protocol: follow in this link

What we did in the lab:
Materials:
• QIAGEN Gel extraction Kit
• Buffer QG
• Gel bands C1 and C2
• Isopropanol
• 2 ml Eppendorfs

Method:
1. Calculate the amount of buffer QG for the extraction :

Table 8
	Weight of our tubes (mg)	Weight of our extraction (mg)	Buffer QG (μl)
C1	1.0360	40.2	121
C2	1.0154	32.8	97

2. Add 1 volume of isopropanol for C2 (32.3 μl) and 3 volumes of isopropanol for C1 (121 μl) 3. Recovery of DNA in 40 μl extraction buffer (20x2 μl)

Results:
Measurement of concentration with a dilution of 1⁄250 :

Table 9
	C1	C2
OD₂₆₀	0.003	0.003
OD₂₈₀	0.003	0.003
[DNA] (μg⁄ml)	30	120

Aim: To link vectors with the inserts.

Protocol: follow in this link

What we did in the lab:
Materials:
• Colonies C1 v2 and C2 v2 Insert bands
• Salt solution
• Distilled water
• TOPO vector

Method:

Table 10
	C1 v2	C2 v2
Colonies (μl)	4	4
Salt solution (μl)	1	1
Distilled water (μl)	5	5
TOPO vector (μl)	1	1
V_final (μl)	6	6

Aim: Save the bacteria with the recombinant vector for storage.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiological equipment
• Carbenicillin 50 μg⁄ml
• LB medium
• 50 ml Falcons
• 2 ml Eppendorfs
• Recombinant vector
• BL21DE3 colonies

Method:
1. Prepare in a falcon of 50 ml a preculture of the recombinant vector with one colony, 5 ml of LB and 5 μl of carbenicillin 50 mg⁄ml
2. Add 5 ml of LB and centrifuge 7 minutes at 3500 RPM
3. Discard the supernatant and add 5 ml of LB. Then, centrifuge again 7 minutes at 3500RPM
4. Discard the supernatant and add 5 ml of LB
5. Put 200 μl of this preparation into another Falcon of 50 ml, add 25 ml of LB and 25 μl of carbenicillin
6. Let the culture shake at 37°C for 4 hours (until the culture growth is sufficient, OD of approx. 0.7 at 600 nm) and check the concentration at several times during it :

Table 11
Time	OD_600nm
0	0.007
1h19	0.059
2h39	0.458
3h54	0.827

7. In 9 Eppendorfs of 2 ml, put 1 ml of LB with AGAR and 1 μl of carbenicillin. Then mix it
8. Dip a toothpick in the culture, and stab in each Eppendorf, and let it incubate at 37°C overnight. Afterwards store at 4°C.
9. On the July 28, 2016 : restreaking of cultures in a Petri dish (verification of STABs)

Aim: To determine if the two primers For (Forward direction) and Rev (Reverse direction) that were previously prepared are specific or not.

Protocol: follow in this link

What we did in the lab:
Materials:
• Mastermix 2X
• Primer S
• Primer AS
• Distilled water
• C1 and C2 inserts
• Phusion DNA polymerase
• Tubes for PCR
• PCR machine

Method:
1. For each inserts prepare the following Globalmix :

Table 12
Volume (μl)	Insert	Primer S	Primer AS
Mastermix 2X	75	75	75
Primer S	3	3	Ø
Primer AS	3	Ø	3
H₂O	65.4	68.4	68.4

2. Spread the previous samples and add DNA to have :

Table 13
Volume (μl)	Insert	Primer S	Primer AS	Without DNA
Globalmix	48.4	48.4	48.4	48.4
C1 or C2	1	1	1	1
Phusion	0.2	0.2	0.2	0.2
Total	50	50	50	50

3. Dispense these samples in 10 tubes and launch the PCR for 24 cycles

Aim: Check the results of the previous PCR of the new versions of C1 and C.

Protocol: follow in this link

What we did in the lab:
Materials:
• Ladder
• Inserts (old)
• C1 and C2
• Primer S
• Buffer EX
• Electrophoresis machine

Method:
Add 5 μl of DNA and 1 μl of lead buffer EX:

Table 14

Samples L1 L2 L3 L4 L5 L6 L7 L8

Contain
Ladder C1 + insert C1 + S C1 + AS C2 + insert C2 + S C2 + AS C without DNA

Results:
None of the bands of our samples can be seen because the temperature is not suitable (too hot). This experiment has to be redone another time at lower temperature.

Figure 1

Aim: PCR of the new versions of C1 and C2 at lower annealing temperature
Re-amplify some of the previous PCR reaction solution with fresh dNTPs, buffer and polymerase..

Protocol: follow in this link

What we did in the lab:
Materials:
• Samples from the previous PCR of the 26⁄07
• Tubes for PCR
• PCR machine
• dNTP
• MgCl₂ solution
• Phusion

Method:
1. Take 7 of the samples from the previous PCR (which contains only 45 μl after the electrophoresis)
2. With these samples, prepare the following samples by adding in order : dNTP, MgCl₂ and Phusion enzyme :

Table 15
Volume (μl)	Insert	Primer S	Primer AS	Without DNA
Samples	45	45	45	45
dNTP	6	6	6	6
MgCl₂	8.4	8.4	8.4	8.4
Phusion	0.5	0.5	0.5	0.5
V_final	60	60	60	60

3. Vortex each samples and centrifuge at room temperature at 3500 RPM
4. Run PCR following previous protocol, except for the lowered temperature.

Aim: Know the concentration of the inserts.

What we did in the lab:
Materials:
See protocol

Result

Table 16
	C1 v2	C2 v2
V_TE (μl)	498	498
V_DNA (μl)	2	2
Ratio	X	X
A₂₆₀	0.002	0.001
A₂₈₀	0.001	0.001
[Insert] (μg⁄ml)	20	25

Aim: Send of the recombinant vector C2.1 (from the Midiprep of the 26⁄07) in sequencing to Eurofins-MWG.

Results:

Table 17
[DNA_C2.1] (ng⁄ml)	450
V_DNA (μl)	3.3
V_H2O (μl)	11.7
V_{primer d1⁄50} (μl)	2
V_total (μl)	17

Aim: Know the concentration of purified C1 (For + Rev).

What we did in the lab :
Materials :
• Ulltrospec 3100
• Quartz cuvette
• TE buffer
• C1 DNA from the PCR purification (27⁄07)
• Petri dishes

Method
1. Do the blank with 500 μl of TE buffer
2. For the sample, use 498 μl of TE buffer and 2 μl of C1 DNA from the PCR purification

Results

Table 18
OD₂₆₀	OD₂₈₀	[DNA_C1] (μg⁄ml)
0.002	0.001	50

“”/ — Aim: Revelation of proteins in each well.

What we did in the lab :
Materials :
• TGS 10X
• PAGE ruler molecular weight marker (Thermofisher)
• Distilled water
• Protein fraction solutions from FPLC
• Buffer A
• Laemli 2X
• SDS PAGE gel, electrophoresis chamber, 3000 V power supply (Biorad)

Method
1. Prepare 400 ml of TGS 1X with 40 ml of TGS 10X and 360 ml of distilled water
2. Dilute protein solutions :

Table 19

Fractions from FPLC Dilution Contain

19, 21, 25 Ø Ø

30, 34, 36 (1⁄100) in buffer A 1μl of protein with 99μl of buffer A

37, 38, 39 (1⁄10) in buffer A 1μl of protein with 9μl of buffer A

3. In each gel well, add 10 μl of diluted protein solution and 10 μl of laemli 2X
4. Put all samples at 95°C during 5 minutes and follow the protocol for SDS PAGE gel

Results :
The distribution of samples in lanes is the same as the previous experiment:
Figure 4 1 : Ladder
2 : Well 19
3 : Well 21
4 : Well 25
5 : Well 30
6 : Well 34
7 : Well 36
8 : Well 37
9 : Well 38
10 :Well 39

Aim: Test the specificity of our two primers : For and Rev.

Protocol: follow in this link

What we did in the lab :
Materials :
• dNTP
• Solution of MgCl₂
• RNA free
• Buffer 10X pfu DNA polymerase
• Inserts C1 and C2
• For primer
• Rev primer
• 2 ml Eppendorfs
• Distilled water

Method
1. Make a global mix for the 7 samples :

Table 20
	Volumes (μl)
dNTP	22.5
MgCl₂	22.5
RNA free	327.6
Buffer 10X pfu	22.5
Total	417.6

2. Distribute the global mix in each tubes and add DNA :

Table 21
Tubes	Name	V(premix) (μl)	V(C1) (μl)	V(C2) (μl)	V(primer For) (μl)	V(primer Rev) (μl)	V(H₂O) (μl)
1	C1 insert For and Rev	46.4	1	0	1	1	0
2	C1 insert For	46.4	1	0	1	0	1
3	C1 insert Rev	46.4	1	0	0	1	1
4	C2 insert For and Rev	46.4	0	1	1	1	0
1	C2 insert For	46.4	0	1	1	0	1
1	C1 insert Rev	46.4	0	1	0	1	1
1	Without DNA	0	0	0	0	0	0

3. Put the tubes in a PCR machine heat to 94°C during 1 minute and then add the enzyme.

Aim: Test the specificity of our two primers : For and Rev.

Protocol: follow in this link

What we did in the lab :
Materials :
• 100 ml Erlenmeyer
• TAE 1X
• Agarose gel
• Ethidium bromide drops (EB)
• Gel electrophoresis apparatus
• Gene ruler ladder
• Loading buffer 6X
• DNA (C1 and C2, For and Rev)

Method
1. Prepare a 0.71% agarose gel :
1.a In an Erlenmeyer of 100 ml, put 50 ml of TAE1 X and add 0.35g of agarose
1.b Warm it in a microwave 2 minutes and let it cool down a little
1.c Add 2 drops of EB and mix it
1.d Transfer it on combs in a gel caster and let it solidify
2. Put samples in the wells:

Table 22

Wells Samples

1
7 μl of gene ruler

2
2 μl of 6X + 10 μl of DNA (C1 For and Rev)

3
2 μl of load 6X + 10 μl of DNA (C1 For)

4
2 μl of load 6X + 10 μl of DNA (C1 Rev)

5
2 μl of load 6X + 10 μl of DNA (C2 For and Rev)

6
2 μl of load 6X + 10 μl of DNA (C2 For)

7
2 μl of load 6X + 10 μl of DNA (C2 Rev)

8
2 μl of load 6X + 10 μl of distilled water

Results :

Figure 5 1 : Gene ruler 7 μl
2 : C1 For + Rev
3 : C1 For
4 : C1 Rev
5 : C2 For + Rev
6 : C2 For
7 : C2 Rev
8 : Without DNA
The experiments did not work.

Aim: Extracting DNA from the colony obtained after transformation.
Protocol: follow in this link

Materials :
• Miniprep QIAGEN Kit
• TOPO cloning colony
• Buffer EB
• Spectrophotometer

Method
1. For the Miniprep prepare 6x 2 ml of cultures and recover it in 50 μl of buffer EB
2. Do the prep with the QIAGEN Miniprep Kit
3. Measure the absorbance of the products to determine the amount of DNA obtained after extraction

Results :

Table 23
Samples	A₂₆₀	A₂₈₀	[DNA] (μg⁄ml)
1	0.009	0.001	120 → wrong
2	0.001	0.003	10
3	0.001	0.003	11

Aim:Check if the insert is in the TOPO vector.
Protocol: follow in this link

Materials :
• Biochemical equipment
• TOPO vector
• Xba I (NEB)
• Hind III (NEB)
• Buffer Cutsmart 10X (NEB)
• Distilled water

Method

Table 24
	Volumes (μl)
DNA (10 μg⁄ml)	5
XbaI	1
HindIII	1
Buffer Cutsmart 10X	4
H₂O	29
Total	40