To increase the amount of plasmid by transformation in competent cells. The amount of plasmid supplied is insufficient to perform all of our future experiments. Therefore we need to amplify the amount of plasmids.
• subcloning competent cells
• pSB1C3 plasmid (from shipped BioBrick-competent cells testing kit), chloramphenicol resistance
• pET43.1a (GE health care), ampicillin resistance (or carbenicillin)
• SOC (Super optimal broth) media
• LB (Luria broth) Agar plates containing 50 µg/ml carbenicillin or 34 µg/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 in volume of 50 µl/vial.
2. Add 50 pg plasmids to each 50 µl of competent cells vial and tap gently.
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 +135 µl 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.
Aim :
To start a culture for Miniprep. 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)
• 25 ml flasks
• Carbenicillin 50 mg/ml
• Chloramphenicol 34 mg/ml
• LB medium
Method:
1.One colony is picked from the plates and shaken in 25 ml of LB supplemented with carbenicillin or chloramphenicol at 50 mg/ml or 34 mg/ml respectively.
2.The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.
Aim:
To perform a midiprep to isolate plasmid DNA of pSB1C3 and pET43.1a(+)
The amplification method to increase the amount of plasmid is called Mini or Midiprep.
The protocol in step 1 ask for spinning at 6000g 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.
1. Use culture from overnight (17 hours) 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 (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 into 10 Eppendorf 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 minutes, 15 000g, at room temperature).
12. Add 3.5 ml of isopropanol, mix to precipitate the DNA.
13. Centrifuge (30 minutes, 15 000g, at room temperature).
14. Remove isopropanol with 1 ml pipet without taking DNA and place into chemical waste container.
15. Add 1 ml of 70% ethanol, centrifuge again (15 minutes, 15 000g, at room temperature) and let air dry.
16. Resuspend in 50 ml of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C.
Aim:
To linearize the different plasmids with appropriate enzymes.
We perform restriction enzyme digestion in order to recover linear backbones of the plasmids. We choose appropriate restriction sites based on the host plasmids.
• Restriction enzymes: Xba I, Hind III, Spe I, BamH I (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer
Method:
Measure the quantity of plasmid using a spectrophotometer (ultrospec 3100 pro, Pharmacia GE health care)
Results:
Table 1 : Absorbances
λ= 260 nm
pSB1C3
pET43.1 a(+)
A260 nm
0.008
0.026
A280 nm
0.001
0.014
A260 nm/A280 nm
4.00
1.85
C diluted
0.4 ng/µl
1.9 ng/µl
C final
26.7 ng/µl
86.7 ng/µl
The concentrations measured after dilution were too low. We eventually switched to a Nanodrop (Thermofisher) because the plastic uvette gave too much background.
Method for digestion by Restriction enzymes
1. Mix all the reagents and let digest during 2 hours at 37°C.
⚠ Big volumes must be added first!
• pET43.1 a at 87.7 ng/µl will be digested by BamH I and Hind III (NEB).
• pSB1C3 at 26.7 ng/µl will be digested by Spe I and Xba I (NEB).
2. We began the digestion 17h20. Here we digest 400 ng of DNA. We doubled Hind III volumes because this enzyme has only 50% of efficiency in Custmart (NEB) buffer.
