Team:Aix-Marseille/Protocols
DH5α competent cells preparation
- The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.
- Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.
- The next day, recover your starters and seed 1/60 bacterial cells in 200 mL of LB, then incubate at 37°C and 180 rpm.
- Keep measuring the OD600. When it's between 0,4 and 0,6, recover your cells an pool them in cold 50 mL Falcon tubes.
- Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
- Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
- Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
- Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.
| Product | For 40 mL |
|---|---|
| KAc (1M) | 1.2 mL |
| MnCl2 (0.5M) | 4 mL |
| KCl (1M) | 4 mL |
| CaCl2 (0.1M) | 4 mL |
| Glycerol (80%) | 7.5 mL |
| H2O | 19.3 mL |
| Product | For 8 mL |
|---|---|
| KCl (1M) | 800 µL |
| MOPS (1M) | 400 µL |
| CaCl2 (0.1M) | 6 mL |
| Glycerol (80%) | 1.5 mL |
| H2O | 500 µL |
Transformation
- Recover the needed number of competent cells tubes, adding an additional negative control tube.
- Add DNA (1µL) into the competent cells tubes.
- Incubate tubes on ice for an hour.
- Incubate tubes at 42°C for 2 minutes for thermal choc.
- Leave tubes on ice for 5 minutes.
- Leave tubes at room temperature for 5 minutes.
- Add 1 mL of LB per tube, then incubate for 1 hour at 37°C and 180 rpm.
- Spread cells on Petri dishes and incubate overnight at 37°C.
Monarch PCR and DNA cleanup Kit
All centrifugation steps ~13 rpm in a typical microcentrifuge. This ensures all traces of buffer are eluted at each step.
- A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. Add 100 μl DNA Cleanup Binding Buffer to the 50 μl sample.
- Insert column into collection tube, load sample onto column and close the cap. Spin for 1 minute, then discard flow-through.
- Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discard flow-through.
- Repeat Step 5. This step is recommended for removal of enzymes that may interfere with downstream applications (e.g., Proteinase K).
- Spin for 1 minute.
- Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
- Add 15μl of hot water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute the DNA.
- Repeat step 6.