• 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Swing bucket centrifuge (JOUAN GR41)
• Colonies of C2 v2 and B1 v2
• carbenicillin at 50 mg⁄ml
• LB medium
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
1. In a 50 ml falcon, put 48 ml of LB and 48 μl of carbenicillin.
2. For B1 v2 :
2.a Prepare 13 Eppendorfs of 1.5 ml in which add 1 ml of the previous mix.
2.b Take with a toothpick colonies on the petri dish.
2.c Place the toothpick in a tube.
2.d Let incubate overnight at 37 °C and 150 rpm.
3. For C2 v2 :
3.a Prepare 20 eppendorfs of 1.5 ml with 1 ml of LB and carbenicillin mix.
3.b Take colonies and place it as previously explained.
3.c Let incubate overnight.
Aim :
As the transformations did not work (B2, E1 and E2 in pET 43.1(a+) ) with TOP 10 competent cells, we take the products of the midiprep done on the 4th of August and we digest before redoing the transformation.
• Restriction enzymes: Xba I, Hind III (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37 °C water bath
• Shaking incubator (INFORS HT)
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
1. Realize a Mastermix and store it on ice :
Table 1: Volumes
Reactants
Volumes (µl)
Xba I
30
Hind III
30
Buffer 2.1
90
Distilled H2O
90
Total
150
2. In For each insert (B2, E1, E2), prepare 10 tubes of 1.5 ml (10 eppendorfs of miniprep products).
3. In each tube, put 25 μl of DNA and 5 μl of Master mix.
4. Let digest 2 hours at 37 °C, then incubate 5 minutes at 65 °C.
Aim :
Check if the digestion has been done, it would mean that the plasmid contains the insert.
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Colonies B2, E1 and E2
• Electrophoresis chamber
• TAE 1X
• Ethidium bromide drops (EB)
Method :
1. Make a gel with 1.4 g of agarose, 200 ml of TAE 1X and 4 droplets of EB.
2. Prepare the samples with 30 µl of digested mix and 6 µl of Gene ruler (Thermofisher)ladder.
3. Follow the deposit table :
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 1.5 ml eppendorfs
• Takara enzyme
• Primers S and AS
• dNTP mix 10 mM
• Tak Ex Buffer 6X
• Distilled H2O
• MgCl2
Method :
1. Prepare the following tubes :
Table 2 : Volumes
Mix with two primers
Mix with primer S only
Mix with primer AS only
Takara enzyme (µl)
6
6
6
Primer S (µl)
6
6
Ø
Primer AS (µl)
6
Ø
6
MgCl2 (µl)
15
15
15
dNTPs (µl)
15
15
15
Buffer (µl)
30
30
30
H2O (µl)
216
222
222
Total (µl)
294
294
294
2. For each mix, spread 49 µl of it in the samples.
3. Add 1 µl of DNA following the number of tubes :
Table 3 : Samples
Mix with two primers
Mix with primer S only
Mix with primer AS only
A1
tube 1
tube 2
tube 3
A2
tube 4
tube 5
tube 6
D1
tube 7
tube 8
tube 9
D2
tube 10
tube 11
tube 12
And tube 13 is the one without DNA
4. Launch the process of PCR.
5. Do an electrophoresis with the results of PCR
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Qiagen Miniprep kit
• Digestion enzyme Xba I and Hind III
• Digestion buffer 2.1
• 1.5 ml Eppendorfs
• Electrophoresis chamber
• Distilled water
Method :
1. Use the Qiagen kit for our cultures from the 8th of August :
13 Eppendorfs of B1.
20 Eppendorfs of C2.
2. Digest the plasmid with the following volumes for each sample :
Table 7 : Volumes
Volumes (µl)
DNA
5
Xba I
1
Hind III
1
Buffer 2.1
2
H2O
11
Total
20
3. Analyze the digestion by electrophoresis on an agarose gel prepared with 1.05 g of agarose in 125 ml of TAE 1X.
4. Launch the electrophoresis, following the deposit table :
&• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Carbenicillin at 50 mg/ml
• Digestion enzyme Xba I and Hind III
• LB medium
• pET43.1(a+)
• C1 v2 colonies
• Shaking incubator (INFORS HT)
Method:
1. Prepare 20 ml of LB with 20 µl of carbenicillin.
2. Put 1 ml of this mix in twenty 1.5 ml Eppendorfs.
3. Take 20 colonies of the petri dish C1 v2 and put them in the previous Eppendorfs.
4. Let incubate overnight at 37°C and 150 rpm.
Aim:
Create a stock of antibiotic.
What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Carbenicillin at 50 mg/ml
• 1.5 ml Eppendorfs
• 15 ml Falcon
• Distilled water
Method:
1. Prep: Put 500 mg of carbenicillin in a 15 ml Falcon and 10 ml of distilled water. Then, put the Falcon on ice.
2. Aliquot the mix in 10 Eppendorfs of 1.5 ml.
3. Store at -20°C
The PCR works properly since we noticed significant bands at the expected level.
Aim: After their ligation in TOPO cloning vector, they will be transformed into TOP 10 cells. Protocol: follow in this link
What we did in the lab : Materials
• Ligation’s products of A1⁄A2⁄D1⁄D2
&bull ; TOP 10 competent cells
&bull ; SOC
• Microbiology equipement
• Xgal at 10 mg⁄ml
Method
1. Add 6 μl of ligation product in 50 μl of competent cells.
2. Put the samples 30 minutes on ice, then 40 seconds at 42 °C.
3. Put the samples 3 minutes on ice.
4. Add 150 μl of SOC.
5. Let incubate 40 minutes at 37 °C and 150 rpm
6. Take LB with carbenicillin petri plate and add Xgal to reach 40 μg⁄ml. As the volume is about 25 ml, add 1 mg of Xgal and as the stock solution is 10 mg⁄ml, spread 100 μl on the plate.
7. Spread bacteria on four distincts petri dishes (one for each insert).
Aim: After their ligation we must transform the inserts into bacteria. Protocol: follow in this link
What we did in the lab : Method
Add 10 μl of ligation product (to have 100 mg) at the beginning.
Aim: Get back the DNA. Protocol: follow in this link
What we did in the lab : Method
We use a final volume of 50 μl
Aim: Split the insert and the plasmid. 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)
• Qiagen Miniprep kit
• Digestion enzyme Xba I and Hind III
• Digestion buffer 2 X
• 1.5 ml Eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)
Method:
1. Realize a master mix with :
Table 8
Volumes (µl)
Xba I
20
Hind III
20
Buffer 2X
40
H2O
220
Total
300
2. Put μl of the master mix in each of the twenty 1.5 ml Eppendorfs and add 5 μl of DNA.
3. Let incubate one hour at 37 °C and 150 rpm, then 5 minutes at 65 °C
Results The 10th of August, we realize that we forgot to put Xgal, so we re-spreaded the previous mix on petri dishes with Xgal.
Aim: We want to produce 5 μ g of dephosphorylated pET 43.1 (a+) from pET 43.1 (a+) at 400 ng⁄ml and we start with the digestion. 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)
• Qiagen Miniprep kit
• Digestion enzyme Xba I and Hind III
• CutSmart buffer
• 1.5 ml Eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)
Method:
1. Put all the following reactants in a 1.5 ml Eppendorf and let digest one hour at 37 °C :
Table 9
Volumes (µl)
DNA
12.5
Xba I
2
Hind III
4
Buffer CutSmart
5
H2O
26.5
Total
50
2. Inactivate the enzymes 5 minutes at 65 °C.
Aim: Get back the digested and purified plasmid before dephosphorylation. Protocol: follow in this link
What we did in the lab: Materials:
• Products from the digestion of pET 43.1 (a+) with Hind III and Xba I
• Agarose
• Electrophoresis chamber Method:
1. Make a 0.7 % agarose gel
2. Prepare the chamber to do the migration at 100 V with two wells for pET43.1(a+) and one for the ladder.
3. Take the results and follow the kit steps of Qiagen extraction kit. Results>
We notice two bands for digested pET43.1(a+), one at 6000 bp and another between 1000 and 1500 bp. We extract the bands at 6000 bp.
We obtained :
m1 = 0.4079 g
m2 = 0 .3720 g
Aim: Check if the digestion works properly and if we have inserts. Protocol: follow in this link
What we did in the lab: Materials:
• Products from the digestion of C1
• Agarose
• Electrophoresis chamber Method:
1. Take the 20 μl of each sample from the digestion and add 4 μl of loading buffer 6X.
2. Do the electrophoresis, following the deposit table :
Ladder | Ø | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | Ø | Ladder
Ladder | Ø | 17 | 18 | 19 | 20 Results
There is no insert. The colonies do not contain our insert, we must redo the experiment with new colonies.
Aim: Transform the bacterias with our recombined plasmid. Protocol: follow in this link
What we did in the lab: Method:
The volumes of insert are too small, we add 5 μl of H2O and diluted at 1⁄100.
We performed a transformation in DH5α with 1 μl of DNA and 50 μl of competent cells.
Aim: Have more antibodies. Results We obtained 30 μl⁄aliquot.
Aim: Increase the quantity of colonies containing inserts. Protocol: follow in this link
Aim: Ligate the insert and the plasmid. Protocol: follow in this link
Aim: Transform our inserts in TOP 10 competent cells. Protocol: follow in this link
Aim: Have bacteria with the right plasmid to produce our protein. Protocol: follow in this link
What we did in the lab: Materials:
• Digested B1 and C2
• BL21DE3 competent cells
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Shaking incubator (INFORS HT)
• SOC Method:
1. Take 3 samples of C2 v2 (10, 13 and 16) and 2 samples of B1 v2 (6 and 8).
2. Put 1 μl of DNA in 99 μl of H2O. Then, put 1 μl of DNA (B1 or C2) in 50 μl of BL21DE3 competent cells.
3. Put the samples 30 minutes on ice and then 40 seconds at 42 °C.
4. Put the samples 3 minutes on ice.
5. Add 150 μl of SOC and let incubate 30 minutes at 37 °C and 150 rpm.
6. Spread the mix on a petri dish with LB and carbenicillin.
7. Let incubate overnight at 37 °C and 150 rpm.
Aim: Have the concentration of digested pET 43.1. Results Measure the concentration of digested pET 43.1(a+) with a Nanodrop. For the first tube, we find a concentration of 6.8 ng⁄μl in 46 μl. For the second tube, we find a concentration of 8.8 ng⁄μl in 46 μl. Then, store the samples at −20 °C.
Aim: Transform C2 v2 and B1 v2 in pET43.1a(+) and DHα . Protocol: follow in this link
Results For B2 (4, 7, 9), E1 (1, 2) and E2 (2, 3, 4, 5, 6, 7) we see small colonies so we let and 25 μl of carbenicillin.
For B1 v2 and C2 v2 in pET43.1(a+), transformations were successful.
For A1, A2 and D1, D2 there were too many bacteria growing so we took a colony, diluted it in LB and then spread it on petri dish with LB, Xgal and carbenicillin.
Aim: To produce proteins. What we did in the lab: Materials:
• Spectrophotometer Ultrospec 3100
• iPTG at 0.5 M
br/> Method:
1. Make two measurements separated of 30 minutes :
Table 10
Sample
1
2
3
4
5
6
Time of addition of iPTG
Concentration (ng⁄μl)
B1 (1)
0.022
0.062
0.152
0.344
0.557
0.694
16 h 55
0.859
B1 (2)
0.185
0.417
0.693
15 h 25
0.956
B1 (3)
0.075
0.211
0.413
0.688
15 h 55
0.920
C2 (1)
0.060
0.166
0.350
0.627
0.699
16 h 25
0.905
C2 (2)
0.044
0.119
0.296
0.510
0.698
16 h 25
0.910
C2 (16)
0.080
0.230
0.445
0.689
15 h 55
0.907
2. When the OD600 nm reached 0.7 keep the last measurement and centrifuge for 3 minutes at 8000 g.
3. Throw away the supernatant and store at −20 °C.
4. Add iPTG to reach 0.3 mM.
Aim: Make the future ligation easier. 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)
• rSAP
• CutSmart buffer
• 1.5 ml Eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)
Method:Method
1. Start with tube 2 (8.6 ng⁄μl in 46 μl) and use the following mix :
Table 11
Volumes ( μl)
DNA
46
CutSmart
6
H2O
6.7
rSAP
1.3
Total
60
2. Let incubate 30 minutes at 37 °C then 5 minutes at 65 °C.
3. Do the same for tube 1.
Aim: Get back the DNA. Protocol: follow in this link
What we did in the lab
What we did in the lab: Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• LB medium
• Carbenicillin at 50 mg⁄ml
• B1⁄E1⁄E2 in TOPO Method
We do 31 precultures with a mix with 1 ml of LB and 1 μl of carbenicillin to have :
3 samples of E1
14 samples of B2
14 samples of E2
Aim: Send our insert for sequencing as the transformations in BL21DE3. Protocol: follow in this link
What we did in the lab
Aim: Have precultures to redo the cultures in 4 x 1 l of LB as they grow well. Protocol: follow in this link
What we did in the lab
Aim: Increase the quantity of plasmid for the next ligation. Protocol: follow in this link
What we did in the lab
Aim: Increase the quantity of DNA. Protocol: follow in this link
What we did in the lab
Aim: Check if the colonies we took contain the insert. Protocol: follow in this link
What we did in the lab
What we did in the lab Materials
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Digestion enzyme Xba I and Hind III
• Digestion buffer 2.1
• 1.5 ml Eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)
• Inserts B2⁄E1⁄E2
Method
1. In a 1.5 ml Eppendorf, put :
Table 12
Volumes (μl)
DNA
5
Xba I
1
H2O
11
Hind III
1
Buffer 2.1
2
Total
20
2. Let incubate one hour at 37 °C , then 5 minutes at −20 °C.
Aim: Produce the protein in higher quantity. 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)
• iPTG
• Carbenicillin at 50 mg⁄ml
Method
1. In a 2 l Erlenmeyer, put 1 l of LB and 1 ml of carbenicillin and incubate at 37 °C and 150 rpm to warm the liquid.
2. For each preculture of 25 ml, put it in a 50 ml Falcon, centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 5 ml of clean LB.
3.Centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 20 ml of clean LB.
4. In each 2 l erlenmeyer, add 5 ml of our preculture : 4 erlenmeyer for C2 and 4 for B1.
5. Let incubate at 37 °C ann 150 rpm and measure the DO.
6. Once the DO reaches 0.7, add 1 ml of iPTG.
7. Let incubate for 3 hours.
8. Centrifuge the cultures.
9. Resuspend the pellet in 10 ml of lysis buffer (See protein purification protocol) in a 50 ml Falcon of known weight.
10. Store at −20 °C.
Aim: Check if the digestion works. Protocol: follow in this link
What we did in the lab Materials
• Agarose
• Qiagen kit
• Nanodrop
• Electrophoresis chamber
• Loading buffer 6X
Method
1. Add 10 μl of loading buffer 6X to reach 50 μl for each sample.
2. Do an electrophoresis and extract the bands of the palsmid digested. We obtaines two tubes :
Tube1 : m1 = 1.276 − 0.9964 = 131.2 mg
Tube2 : m2 = 1.1524 − 0.9994 = 153.0 mg
3. Follow the Qiagen kit steps for a final volum of 50 μl.
4. Measure the concentration with the Nanodrop to see the results.
5. Store at −20 °C.
