Team:Pasteur Paris/Microbiology week11

Aim: To perform a Miniprep to isolate pET43.1a(+) plasmid DNA with the inserts B1 col1, B1 col2 and C2 col1. The amplification method to increase the amount of plasmid is called Miniprep.

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

Method:
The protocol in step 1 asks for spinning at 6000g but we can only achieve 3500g so we used 3500g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.
1.Follow QIAGEN kit steps

Aim: To get back our insert from the Miniprep of our inserts with appropriate enzymes.
We perform restriction enzyme digestion in order to recover our inserts. We choose appropriate restriction sites based on the host plasmid.

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: Xba I, Hind III (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer
Method:
1. Mix all the reagents and let digest during 2 hr at 37°C.
Big volumes must be added first!
Beginning of digestion 12h10.

Reactants	A1	D1
Vol_DNA	20 µL	20 µL
Vol_{Xba I}	1 µL	1 µL
Vol_{Hind III}	1 µL	1 µL
Vol_H2O	5 µL	5 µL
Vol_{Buffer Cutsmart (10X)}	3 µL	3 µL
Vol_total	30 µL	30 µL

Volumes

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Store at -20°C

Aim: This step check the digestion efficiency. Moreover, the inserts will be purified during this step because they will be extracted from the gel.

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: Xba I, Hind III (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer

Method:
• Electrophoresis chamber
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye 6X
• Agarose
• UV table
• Ethidium bromide drops (EB)

Beginning of the electrophoresis at 14h30 at 100 V.

Results:
The gel reveals that A1 contains the insert but the amount of DNA is too low so we will redo the experiment.

Aim: To start a culture for Midiprep.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight in a larger volume of LB media.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipement
• 25 ml flasks
• Carbenicillin 50 mg/ml
• Chloramphenicol 34 mg/ml
• LB medium

Method:
2.One colony is picked from the plates and shaken in 25 ml of LB supplemented with carbenicillin at 50 µg/ml. This step is done with the inserts A1/A2/D1/D2 and B1/B2 for sequencing.
3.The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Aim: To perform a Midiprep to isolate plasmid DNA of pET43.1a(+) with A1/A2/D1/D2 and B1/B2 for sequencing.
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)
• QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)

Method:
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500g so we used 3500g for 20 minutes. We will follow most of the protocol of QIAGEN Midiprep 2016 except for a few modifications, which we describe, therefore, below.
1. Use culture from overnight (17 hr) step on June 7, 2016
2. Pour culture in 50 ml Falcon and centrifuge (15 min, 3500g, 4°C)
3. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet
4. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue.
5. Prepare syringes with their cap and the reservoir (on 50 ml Falcon)
6. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white.
7. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer)
8. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer
9. Prepare 10 tubes of 2 ml to aliquot pET43.1a(+) and pSB1C3. Because we have only bench microfuges, we need to dispense our volume in smaller fractions.
10. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes
11. Centrifuge (30 min, 15000g, room temperature)
12. Add 3.5 ml of isopropanol, mix to precipitate the DNA
13. Centrifuge (30 min, 15 000g, at RT)
14. Remove isopropanol with pipet without taking DNA and place into chemical waste container
15. Add 1 ml of 70% ethanol, centrifuge again (15 min, 15 000 g, RT) and let air dry. 16. Resuspend in 50 µl of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C.

Aim: The previous purification shows a significant band at 30 kDa for the samples 22 and 24 but also one at 70 kDa. It probably left some NusA in our column so, it will be cleaned.br>
Protocol: follow in this link

What we did in the lab:
Materials:
• Fast Purification Liquid Chromatography
• Chaotropic reagent (Guanidinium 6M)
• EDTA 0.1 M
• PMSF (100 mM)
• Ni 2+ solution (100 mM)
• Centrifuge (labo Deshmukh)

Method:
1. Melt the pellet of bacteria C2 (from 1 l culture) and resuspend it with 10 ml of buffer A
2. Put the column off the FPLC and wash it with 20 ml of MilliQ water thanks to a finger tight connector and a syringe.
3. Add 20 ml of 6M Guanidinium Hydrochloride chaotropic reagent to denature the proteins fixed to the column
4. Wash the column with 20 ml of water
5. Add 10 ml of EDTA to clean it of nickel ions
6. Wash with 20 ml of water
7. Add 5 ml of NiCl₂ solution to charge the column. The column turns green.
8. Wash with 20 ml of water
9. Sonicate the sample three times one minute at 60%, wait 90 seconds between each sonication, Finally, the sample volume is 40 ml, add 40 µl of PMSF to avoid protein degradation by proteases.
10. Centrifuge 25 min at 16000g (Beckman rotor JA 25.50)
11. Inject the sample on the column of the FPLC
12. Save several stages as frozen samples for later analysis:
• 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 the FPLC in order to know if it is our protein

What we did in the lab:
Materials:
Materials:
• SDS-PAGE chamber
• SDS-PAGE 4-15 % gradient gel (BIORAD)
• Protein migration buffer
• Protein PAGE ruler plsu (Thermofisher)Ladder
• Laemmli 2X
• Gel code blue (Coomassie Blue G250)
• Microbiology equipment (Follow this link)

Method:
1. In 9 1.5 ml eppendorf, put 20 µl of a sample and 20 µl of Laemmli 2X.
2. Place the gel into the cuve and fill it with migration buffer
3. Follow the next deposit table:
• Protein ruler 8ul
• Pellet
• Supernatant
• Wash
• Flow through
• Fraction 13
• Fraction 16
• Fraction 20
• Fraction 22
• Fraction 24
4. Launch the migration at 120V (start at 12h10 and stop at 14h10).
5. Wash the gel three times with distilled water during 5min.
6. Color the gel with Coomassie Blue diluted 1/5 during 30min.
7. Wash with distilled water for 5min then let wash 15min.

Method:
We notice a 30kDa band in the well 9 and 10 so we redo a gel with the fractions 21 to 25. Follow exactly the same protocol but with 30 µl of DNA and 30 µl of Laemmli 2X.
Deposit table:
- Protein ruler 8 µl
- Fraction 19
- Fraction 20
- Fraction 21
- Fraction 22
- Fraction 23
- Fraction 24
- Fraction 25
- Fraction 26
- Fraction 27
We notice a 30kDa band in the fractions 19 to 21 that may correspond to our protein and a 70 kDa band due to NusA in fractions 23 to 25.

Aim: To start a culture for Miniprep.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.

Protocol: follow in this link

What we did in the lab:
Materials:
Materials:
• Microbiology equipement
• 15 ml Falcon tube
• Carbenicillin 50 mg/ml
• LB medium

Method:
17. One colony is picked from the plates and shaken in 3 ml of LB supplemented with carbenicillin at 50 µg/ml. This step is done with the inserts A1/A2/D1/D2/C2 and two colonies B1.
18. The Falcon tube is placed in a shaking incubator at 37°C, 150 rpm overnight.

Aim: To perform a Miniprep to isolate plasmid DNA of pET43.1a(+) with the inserts A1 (4 tubes) / B1(2 tubes) / C2 (1 tube) / D1 (2 tubes) and D2 (1 tube).
The amplification method to increase the amount of plasmid is called Miniprep.

Protocol: follow in this link

What we did in the lab:
Materials:
• 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 3500g so we used 3500g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.
19. Follow QIAGEN kit steps

Aim: Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)

What we did in the lab:
Materials:
• Nanodrop (Thermofisher)
• Elution buffer from QIAGEN kit
• Microbiology equipment (Follow this link)

Method:
Analyze absorbance at 260 nm
1. Clean the Nanodrop with water
2. Make the blank with 1 µl of elution buffer
3. Put 1 &181;l of your sample on the Nanodrop
4. Make the measure and clean the Nanodrop between each measure

Results:

λ= 260 nm	B1(1)	B1(2)	C2>th>
A_DNA	0.725	0.741	0.761
C final	36.3 ng/µl	37.0 ng/µl	38.0 ngµl

Absorbance

5. Preparation of the samples for sequencing: put 15 µl of DNA and add 2 µl of fitted primers
6. Send the samples for sequencing

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.

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: Xba I, Hind III (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer

Method:
4. Mix all the reagents and let digest during 2 hr at 37°C
Big volumes must be added first!
Beginning of digestion 11h45.

Reactants	Each sample
Vol_DNA	30 µl
Vol_XbaI	1 µl
Vol_HindIII	1 µl
Vol_H2O	13 µL
Vol_{Buffer Cutsmart (10X)}	5 µl
Vol_total	50 µl

Volumes

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Store at -20°C

Aim: This step check the digestion efficiency of A1 (4 tubes)/D1 (2 tubes)/ D2 (1 tube). Moreover, the inserts will be purified during this step because they will be extracted from the gel.
Protocol: follow in this link

What we did in the lab:
Materials:
• Electrophoresis cuve
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye 6X
• Agarose
• UV table
• BET

Method:
Each well will contain 30 µl of DNA and 6 µl of Loading Dye.
Follow the next deposit table :
Loading dye (6 µL ) / A1 (1) / A1 (2) / A1 (3) / A1 (4) / D1 (1) / D1 (2) / D2 (2)
Beginning of the electrophoresis at 14h20 at 100 V.

Results:
The gel reveals that A1 (1) and (2) contains the insert but the amount of DNA is too high to be purified. The other clones do not have an insert.

Aim: To start a culture for Midiprep of insert C2 and B1.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.
Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipement
• 25 ml flasks
• Carbenicillin 50 mg/ml
• LB medium

Method:
5. One colony is picked from the plates and shaken in 25 ml of LB supplemented with Carbenicillin at 50 µg/ml.
The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Aim: To perform a midiprep to isolate plasmid DNA pET43.1a(+) with the inserts C2 and B1.
The amplification method to increase the amount of plasmid is called Mini or Midiprep.
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)
• QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)

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 20 minutes. We will follow most of the protocol of QIAGEN Midiprep 2016 except for a few modifications, which we describe, therefore, below.
6. Use culture from overnight (17 hr) step on June 7, 2016
7. Pour culture in 50 ml Falcon nd centrifuge (15 min, 3500 g, 4°C)
8. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet
9. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue.
10. Prepare syringes with their cap and the reservoir (on 50 ml Falcon)
11. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white.
12. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer)
13. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer
14. Prepare 10 tubes of 2 ml to aliquot pET43.1(+) and pSB1C3.
Because we have only bench microfuges, we need to dispense our volume in smaller fractions.
1. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes
2. Centrifuge (30 min, 15000g, room temperature)
3. Add 3.5 ml of isopropanol, mix to precipitate the DNA
4. Centrifuge (30 min, 15 000g, at RT)
5. Remove isopropanol with pipet without taking DNA and place into chemical waste container
6. Add 1 ml of 70% ethanol, centrifuge again (15 min, 15 000g, RT) and let air dry
7. Resuspend in 50 µl of Tris 10 mM pH 8.0, EDTA 1 mM (TE) and store at -20°C

Aim: Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)

What we did in the lab:
Materials:
• Nanodrop (Thermofisher)
• Elution buffer from QIAGEN kit
• Microbiology equipment (Follow this link)

Method:
Analyze absorbance at 260 nm
15. Clean the Nanodrop with water
16. Make the blank with 1 µl of elution buffer
17. Put 1ul of your sample on the Nanodrop
18. Make the measure and clean the Nanodrop between each measure

Results:

λ= 260 nm	B1(a)	B1(b)	C2(a)	C2(b)
A_{260 nm}	1.057/td>	1.323	0.971	0.148
A_{280 nm}	0.627	0.698	0.571	0.104
A_{260 nm}/A_{280 nm}	1.69	1.89	1.70	1.43
C final	52.8 ng/µl	66.2 ngµl	48.6 ng/µl	7.4 ng/µl

Absorbance

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.

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: Xba I, Hind III (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer

Method:
19. Mix all the reagents and let digest during 2 hr at 37°C
Big volumes must be added first! Make a global mix to be more accurate

Reactants	Each sample	Global mix
Vol_DNA	45 µl	0 µl
Vol_{Xba I}	2.25 µl	65.25 µl
Vol_{Hind III}	2.25 µl	65.25 µl
Vol_H2O	1.125 µl	32.65 µl
Vol_{Buffer Cutsmart (10X)}	5.63 µl	163.12 µl
Vol_total	56 /µl	326.27 /µl

Volumes

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Store at -20°C

Aim:This step check the digestion efficiency of E1(3 tubes) / E2 (12 tubes) / B2(14 tubes). Moreover, the inserts will be purified during this step because they will be extracted from the gel.

Protocol: follow in this link

What we did in the lab:
Materials:

• Electrophoresis cuve
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye 6X
• Agarose
• UV table
• BET

Method:
Each well can contain 40 µl so we made two big gels with 20x2 lines on each.
Each sample will contain 62 µl as we add 7 µl of loading dye. They will be divided into two wells.
Deposit table (/// means EMPTY to make the cut easier)
Gel 1 Lane 1 :
Loading dye /// B2(2) / B2(2) /// B2(3) / B2(3) /// B2(4) / B2(4) /// B2(5) / B2(5) /// B2(1) / B2(1) /// B2(6) / B2(6)

Gel 1 Lane 2 :
Loading dye /// B2(7) / B2(7) /// B2(8) / B2(8) /// B2(9) / B2(9) /// B2(10) / B2(10) /// B2(11) / B2(11) /// B2(12) / B2(12)

Gel 2 Lane 1 :
Loading dye /// E1(1) / E1(1) /// E1(2) / E1(2) /// E1(3) / E1(3) /// E2(1) / E2(1) /// E2(2) / E2(2) /// E2(3) / E2(3) /// E2(4) / E2(4) /// E2(5) / E2(5) /// E2(6) / E2(6) /// E2(7) / E2(7)

Gel 2 Lane 2 :
Loading dye /// E2(8) / E2(8) /// E2(9) / E2(9) /// E2(10) / E2(10) /// E2(11) / E2(11) /// E2(12) / E2(12) /// E2(13) / E2(13) /// B2(13) / B2(13) /// B2(14) / B2(14) ///

Aim:To start a culture for Miniprep of insert A1, A2, D1 and D2.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipement
• 25 ml flasks
• 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. 10 colonies are taken from each insert.
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Start incubation at 16h30

Aim:
To start a culture for Miniprep of insert A1, A2, D1 and D2.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipement
• 25 ml flasks
• 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 &#181g/ml. 10 colonies are taken from each insert.
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Start incubation at 16h30

Team:Pasteur Paris/Microbiology week11

click on the weeks

Microbiology Notebook

Week 11

August 16, 2016:

188. SDS-PAGE gel with inserts C2 v2 and B1 v2

189. Harvest the culture of Miniprep

August 17, 2016:

190. Miniprep of precultures B1 col1/B1 col2 and C2 col1

191. Digestion of the plasmid pET43.1a(+) with A1/A2/D1/D2

192. Electrophoresis on agarose gel of digestion products A1/A2/D1/D2

193. Harvest the culture with Midiprep A1/A2/D1/D2/B1 col1/B1 col2/C2

194. FPLC Purification of the protein

August 18, 2016:

195. Miniprep of cultures A1/A2/D1/D2/B1 col1/B2col2/C2

196. Protein gel on SDS-PAGE

197. New colonies of B1 and C2 from 11/08

198. Harvest the culture with Miniprep 4 colonies from A1, A2, D1 and D2, 2 colonies of B1 and 1 colony of C2

199. FPLC Purification of the protein

August 19, 2016:

200. Miniprep of cultures made on the 18/08

201. Measurement of the amount of DNA extracted from the Miniprep of B1 v2 and C2 v2

202. Digestion of the plasmid pET43.1a(+) with A1(0)/A1(1)/A1(3)/A1(4)/D1(3)/D1(4)/D2(2)

203. Electrophoresis on agarose gel of digestion products

204. Sequencing of B1(1)/B1(2)/C2 from 18/08

205. Storage of proteins