To perform a gel extraction to isolate insert DNA purified from its plasmid thanks to the migration. We use the gel made before with inserts B2/E1/E2 but we only extract B2 bands.
• Scalpel
• 2 ml eppendorfs
• Balance
• UV table
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• QIAGEN Gel Extraction Kit
Method:
⚠ Be aware of the risks! UV light burns the eyes and skin so make sure you have the right protection
We used the following gel (with different exposures) :
Figure 21 : picture of the gel with weak exposure
Figure 22 : picture of the gel with hight exposure
Figure 23 :picture of the gel with overexposure
Follow QIAGEN Kit steps according to the next tables for the volumes of QG buffer.
Table 130
Bands
Mass of gel (mg)
Volume of QG buffer (µl)
Band1
432
1296
Band2
423
1269
Band3
450
1350
Band4
426
1278
Band5
315
945
Band6
324
972
Band7
543
1629
Band8
483
1449
Band9
501
1503
Band10
255
765
Band11
312
936
Band12
372
1116
Band13
393
1179
Band14
414
1242
Band15
480
1440
Band16
579
1737
Band17
501
1503
Band18
501
1503
Band19
480
1440
Aim:
Measure the quantity of plasmid using a Nanodrop (Thermofisher).
What we did in the lab:
Materials :
• Nanodrop (Thermofisher)
• Elution buffer from QIAGEN kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
1. Analyze absorbance at 260nm.
2. Clean the Nanodrop with water.
3. Make the blank with 1 µl of elution buffer.
4. Put 1 µl of your sample on the Nanodrop.
5. Make the measure and clean the Nanodrop between each measure.
Results :
Table 131
lambda=260nm
Concentration (ng/µl)
C1
3.5
C2
2.9
C3
3.4
C4
4.2
C5
4.1
C6
15.1
C7
5.9
C8
4.9
C9
4.0
C10
4.9
C11
4.3
C12
4.1
C13
7.2
C14
4.9
C15
4.7
C16
8.5
C17
4.4
C18
3.6
C19
5.2
Aim :
To increase the amount of plasmid by transformation in competent cells.
The amount of plasmid supplied is insufficient to perform all our future experiments. Therefore, we need to amplify the amount of plasmids.
• subcloning competent cells TOP10
• pET43.1(a+) (GE health care), ampicillin resistance (or carbenicillin) ligated with the inserts
• SOC (Super optimal Broth) media
• LB (lysogeny Luria broth) Agar plates containing 50 mg/ml carbenicillin or 34 mg/ml chloramphenicol
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method:
1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells for 50 µl/vial.
2. Add 50 pg plasmids to each 50 µl of competent cells vial and tap gently. Here, add 12.5µ of DNA.
3. Place on ice for 30 minutes. Meanwhile, warm LB agar plates from cold room in 37°C non shaking incubator.
4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 minutes.
5. Add 500 µl of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).
6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.
7. Near Bunsen burner flame, add 45 µl of competent cells +135µl of SOC or 200 µl aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.
8. Place plates inverted in the static 37°C incubator overnight.
Results:
The transformation works we had a lot of colonies, we stored them at 4°C to stop their growth.
Aim :
Purifying the protein produced by BL21(DE3) using a Fast Protein Liquid Chromatography. We pool two pellets of bacteria produced in 1 l of culture each.
• Fast Protein Liquid Chromatography
• Chaotropic reagent (Guanidinium 6 M)
• EDTA 0,1 M
• PMSF (100 mM)
• Ni2+ solution (100 mM)
• Centrifuge (from Deshmukh Gopaul 's laboratory)
• Buffer A (50 mM Tris, 150mM of NaCl)
• Buffer B (50 mM Tris, 150 mM of NaCl, 250 mM Imidazole, pH=7.4)
Method:
1. Melt the pellet of bacteria C2 (from 1 l culture) and resuspend it with 10 ml of buffer A. We had 25 ml of pellet we complete until 40 ml with buffer A.
2. Add 40 µl of PMSF.
3. Put the column off the FPLC and wash it with 20 ml of milliQ water thanks to a fingerpit ans a syringue.
4. Add 20 ml of chaotropic reagent to denaturate the proteins fixed to the column.
5. Wash the column with 20 ml of water.
6. Add 10 ml of EDTA to clean it from nickel.
7. Wash with 20 ml of water.
8. Add 5 ml of Ni solution to charge the column. The column turns green.
9. Wash with 20 ml of water.
10. Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication.
11. Finally, the sample is 40 ml, add 40 µl of PMSF to avoid protein denaturation.
12. Centrifuge 25 minutes at 16000g (rotor JA 25.50).
13. Inject your sample in the FPLC.
14. Get back several samples :
→ C: Crude extract : before centrigugation
→ P: Pellet
→ SN: Supernatant
→ F: Flow through (unfixed proteins)
→ W: Wash 5% of buffer B
→ Fractions (depending on the gradient)
Aim:
Get the size of the protein purified thanks to FPLC in order to know if it is our protein.
What we did in the lab:
Materials:
• SDS-Page cuve
• SDS-Page gel (BIORAD)
• Protein migration buffer
• Protein ladder
• Laemmli 2X
• Coomassie Blue
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method:
1. In nine 1.5 ml eppendorf, put 20 µl of a sample and 20 µl of Laemmli 2X.
2. Let denaturate the proteins 5 minutes at 95°C.
3. Place the gel into the cuve and fill it with migration buffer.
4. Follow the next deposit table :
Protein ruler 8 µl | Crude extract | Supernatant | Pellet | Wash | Fraction 11 | Fraction 13 | Fraction 15 | Fraction 18 | Fraction 19 | Fraction 20 | Fraction 21
4. Launch the migration at 130 V.
5. Wash the gel three times with distilled water during 5 minutes.
6. Color the gel with Coomassie Blue diluted 1/5 during 30 minutes.
7. Wash with distilled water for 5min then let wash 15min.
Aim :
To perform a Miniprep to isolate plasmid DNA of pET43.1(a+) with the inserts A1/A2 and D1/D2 from cultures made on the 22/08. The amplification method to increase the amount of plasmid is called Miniprep. Only 25 colonies grow.
• 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Swing bucket centrifuge (JOUAN GR41)
• QIAGEN Miniprep kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method:
The protocol in step 1 ask for spinning at 6000 g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.
Follow QIAGEN kit steps
Aim:
To get back our insert from the Miniprep with appropriate enzymes. We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.
A1(7 tubes) / A2 (6 tubes) / D1(6 tubes) / D2(6 tubes)
• Restriction enzymes: XbaI, HindIII (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer
Method:
1. Mix all the reagents and let digest during 2 hours at 37°C.
Big volumes must be added first!
2. Make a global mix to be more accurate as we have 25 tubes.
3. Beginning of digestion 12:00AM.
Table 132
Reactants
Each sample
Global mix
VolDNA
25 µl
0 µl
VolXbaI
1 µl
25 µl
VolHindIII
1 µl
25 µl
VolH2O
0 µl
0 µl
VolBuffer 2.1
3 µl
75 µl
Voltotal
30 µl
125 µl
4. Incubate 10 minutes at 65°C to inactivate the enzymes.
5. Store at -20°C.
Aim:
This step check the digestion efficiency of A1(7 tubes) / A2 (6 tubes) / D1(6 tubes) / D2(6 tubes). Moreover, the inserts will be purified during this step because they will be extracted from the gel.
Each well can contain 40 µl so we made a big gels with 20x2 lines. Each sample will contain 36 µl as we add 6 µl of loading dye.
Follow the deposit table :
To start a culture for Miniprep of insert E1 and E2 from cells transformed on the 22/08.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 1 ml eppendorfs
• Carbenicillin 50 mg/ml
• LB medium
Method :
1. One colony is picked from the plates and shaken in 1.0 ml of LB supplemented with Carbenicillin at 50 µg/ml.
2. 20 colonies are taken from each insert.
3. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.
Aim :
To recircle the dephosphorylated plasmid pET43.1(a+) with the insert before the transformation in competent cells.
• Ligation enzymes: T4 ligase (New England Biolabs, NEB)
• Ligation buffer 10X
• 65°C heat table
• 100ng of pET43.1(a+) plasmid (6.8ng/µl)
• 50ng of purified insert B2 (15.8ng/µ)
Method :
Mix all the reagents and let digest during 30 min at room temperature.
&9888; Big volumes must be added first!
Table 133
Reactants
B2
pET43.1(a+)
Volplasmid DNA
14.5 µl
14.5 µl
VolInsert
3.2 µl
0 µl
Volligation buffer
3.7 µl
3.7 µl
VolH2O
14.5 µl
17.7 µl
VolT4 ligase
1 µl
1 µl
Voltotal
36.9 µl
36.9 µl
2. Incubate 5 minutes at 65°C to inactivate the enzymes.
3. Store at -20°C.
Aim:
To increase the amount of plasmid by transformation in competent cells.
The amount of plasmid supplied is insufficient to perform all our future experiments. Therefore, we need to amplify the amount of plasmids.
• subcloning competent cells TOP10
• pET43.1a (GE health care), ampicillin resistance (or carbenicillin) ligated with the inserts
• SOC (Super optimal Broth) media
• LB (lysogeny Luria broth) Agar plates containing 50 g/ml carbenicillin or 34 g/ml chloramphenicol
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells for 50 µl/vial.
2. Add 50 pg plasmids to each 50 µl of competent cells vial and tap gently. Here, add 12.5 µl of DNA.
3. Place on ice for 30 min. Meanwhile, warm LB agar plates from cold room in 37°C non shaking incubator.
4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 minutes.
5. Add 500 µl of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).
6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.
7. Near Bunsen burner flame, add 45 µl of competent cells +135µl of SOC or 200 µl aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.
8. Place plates inverted in the static 37°C incubator overnight.
Results:
The transformation works we had a lot of colonies, we stored them at 4°C to stop their growth.
Aim :
We realize that our column was made of sepharose, a material similar to cellulose and our protein might be retained.
We decided to clean the column with urea.
What we did in the lab :
Materials :
• Urea (6 M)
• Buffer A (50 mM Tris, 150 mM of NaCl)
• 50 ml Falcon flasks
• Dialysis MNCO membrane
• Columns used for the purification
• Syringues
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
1. Inject 5 ml of urea solution with a syringue in the column used to get back Prep1. Get back the flow throught in a Falcon and let denaturate for 5 minutes.
2. Inject 5 ml of urea in the column and get back the flow throught in the Falcon and 10 ml of products.
3. Dialyse the content of the Falcon with the membrane MNCO, 3.500, soaking in 500 ml of buffer A overnight at 4°C.
4. Do the same protocol with Prep 2.
5. Inject 5 ml of buffer A in each column to clean them of urea.
6. Store at room temperature samples before dialysis and after dialysis.
Aim :
To get back our insert from the Miniprep with appropriate enzymes.
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.
C1(10 tubes)
the digestion works since we can clearly notice two distincts bands in each well. We will redo this gel to purify the inserts with all the DNA we have (refer on the 25/08)
Aim :
Get the size of the protein purified thanks to FPLC and dialysis in order to know if it is our protein.
What we did in the lab :
Materials :
• SDS-Page cuve
• SDS-Page gel (BIORAD)
• Protein migration buffer
• Protein ladder
• Laemmli 2X
• Coomassie Blue
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method:
1. In nine 1.5 ml eppendorf, put 10 µl of a sample and 10 µl of Laemmli 2X.
2. Let denaturate the proteins 5 minutes at 95°C.
3. Place the gel into the cuve and fill it with migration buffer.
4. Follow the next deposit table :
Protein ruler 8 µl | Ø | Prep 1 non dialysed | Prep 2 non dialysed | Ø | Prep 1 dialysed | Prep 2 dialysed | Ø | Buffer A used for the dialysis | Ø | Column wash | Ø
4. Launch the migration at 130 V. Start of the migration 4:10PM.
5. Wash the gel three times with distilled water during 5min.
6. Color the gel with Coomassie Blue diluted 1/5 during 30 minutes.
7. Wash with distilled water for 5min then let wash 15 minutes.
Results :
We obtained the following SDS PAGE gel :
Figure 24 : SDS PAGE gel of proteins
Aim :
To perform a Miniprep to isolate plasmid DNA of pET43.1(a+) with the inserts E1/E2 from cultures made on the 23/08. The amplification method to increase the amount of plasmid is called Miniprep.
E1 (19 tubes, one did not grow) / E2 (20 tubes)
• 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Swing bucket centrifuge (JOUAN GR41)
• QIAGEN Miniprep kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.
Follow QIAGEN kit steps
Aim :
To get back our insert from the Miniprep with appropriate enzymes.
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.
E1 (19 tubes, one did not grow) / E2 (20 tubes)
• Four 2 l erlenmeyers
• LB (lysogeny Luria broth)
• IPTG (0.1 M)
• Precultures in 25 ml erlenmeyers
• UV spectrophotometer (Ultrospec 3100)
• Shaking incubator (INFORS HT)
• Centrifuge
• Buffer A (50 mM Tris, 150 mM of NaCl)
Method :
1. Put 1 l of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150 rpm.
2. Once warmed, add 5 ml of preculture in each erlenmeyer.
3. Let grow in the shaking incubator. Start of growth at 11:10AM.
4. Measure the absorbance with the UV spectrophotometer every 30 minutes :
Table 136
Times
C2 (1)
C2 (2)
C2 (3)
C2 (4)
2:22PM
0.113
0.132
0.203
0.143
2:55PM
0.303
0.339
0.421
0.328
3:38PM
0.476
0.509
0.593
0.494
3:52PM
0.614
0.659
0.683
0.609
5. Add IPTG to reach a concentration of 0.1 mM.
6. The last measure before induction is pelleted 3 minutes at 8000 g and stored at -20°C.
7. Let induce overnight in the shaking incubator
8. The day after, measure the OD and store the measure pelleted at -20°C. We measure 1.195.
9. Centrifuge at 4500 rpm the culture and throw out the supernatant, the pellet resuspended in 5 ml of buffer A are stored at -80°C before protein extraction.
Aim :
This step check the digestion efficiency of E1 and E2. E1 (19 tubes, one did not grow) / E2 (20 tubes)
Moreover, the inserts will be purified during this step because they will be extracted from the gel.
Make two big gels with 2x20 wells and a small gel with 12 wells. Each sample will contain 60 µl as we add 10 µl of loading dye to the 50 µl of DNA.
Follow the deposit table :
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 15 ml Falcons
• Carbenicillin 50 mg/ml
• LB medium
Method :
1. One colony is picked from the plates and shaken in 5.0 ml of LB supplemented with Carbenicillin at 50 µg/ml and 4 colonies are taken from each insert.
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.
Aim :
To perform a Miniprep to isolate plasmid DNA of pET43.1(a+) with the inserts A1/A2 and D1/D2 from cultures made on the 25/08. The amplification method to increase the amount of plasmid is called Miniprep. Only 25 colonies grow.
• 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Swing bucket centrifuge (JOUAN GR41)
• QIAGEN Miniprep kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
The protocol in step 1 ask for spinning at 6000 g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.
Colonies that grow are : A1 (1), A1 (2), A1 (3), A2 (1), A2 (2), A2 (3), A2 (4), D1 (2), D2 (1), D2 (2), D2 (3), D2 (4),
Aim:
Measure the quantity of plasmid using a Nanodrop (Thermofisher).
What we did in the lab :
Materials :
• Nanodrop (Thermofisher)
• Elution buffer from QIAGEN kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
Method :
1. Analyze absorbance at 260nm.
2. Clean the Nanodrop with water.
3. Make the blank with 1 µl of elution buffer.
4. Put 1 µl of your sample on the Nanodrop.
5. Make the measure and clean the Nanodrop between each measure.
Results :
Table 137
Absorbance at 260nm (diluted 1/10)
A260
A280
A260/280
Concentration (ng/µl)
A1 (1)
0.393
0.206
1.91
19.6
A1 (2)
0.460
0.245
1.87
23.0
A1 (3)non diluted
1.593
0.850
1.87
79.7
A2 (1)
0.381
0.219
1.74
19.1
A2 (2)
0.303
0.150
2.02
15.1
A2 (3)non diluted
0.211
0.111
2.0
11.1
A2 (4)
0.280
0.158
1.78
14.0
D1 (2)non diluted
0.274
0.148
1.85
13.7
D2 (1)
1.740
0.911
1.91
87.0
D2 (2)
0.274
0.151
1.82
13.7
D2 (3)non diluted
0.214
0.128
1.67
10.7
D2 (4)
0.393
0.256
1.55
19.6
Aim :
To get back our insert from the Miniprep with appropriate enzymes.
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.
A1(3 tubes) / A2 (4 tubes)
• Restriction enzymes: HindIII (New England Biolabs, NEB)
• Restriction enzyme + buffer : XbaI Remix (New England Biolabs, NEB)
• 37°C water bath
• UV spectrophotometer
Method:
1. Mix all the reagents and let digest during 2 hours at 37°C.
⚠ Big volumes must be added first!
2. Make a global mix to be more accurate as we have 25 tubes.
3. Beginning of digestion at 12:15AM.
Table 138
Reactants
Each sample
Global mix
VolDNA
25 µl
0 µl
VolXbaI Remix
5 µl
35 µl
VolHindIII
4 µl
28µl
VolH2O
16 µl
112 µl
Voltotal
50 µl
175 µl
2. Incubate 10 min at 65°C to inactivate the enzymes.
Aim:
This step check the digestion efficiency of A1(3 tubes) / A2 (4 tubes). Moreover, the inserts will be purified during this step because they will be extracted from the gel.
We also depose inserts D1 and D2 undigested to check.
1. Each well can contain 40 µl so we made a big gel with 20x2 lines. Each sample will contain 36 µl as we add 6 µl of loading dye.
2. Follow the deposit table :
All the digestion have worked except A1 (3) that seems to be contaminated. Moreover, D2 (2) and D2 (3) seems to have the insert so we will have to digest them.
Aim:
To perform a gel extraction to isolate insert DNA purified from its plasmid thanks to the migration. We use the gel made before with inserts B2/E1/E2 but we only extract B2 bands.
