Team:Aix-Marseille/Protocols
Contents
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 ~13000 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 a column into a collection tube, load sample onto the column and close the cap. Spin for 1 minute, then discard flow-through.
- Re-insert column into the 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.
- 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 15 μL 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.
Monarch miniprep kit
All centrifugation steps ~13000 rpm in a typical microcentrifuge. If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve. Store Plasmid Neutralization Buffer (B3) at 4°C after opening.
- Pellet 1–5 mL bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
Note: For a standard miniprep to prepare DNA for restriction digestion or PCR, we recommend 1.5 mL of culture, as this is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).
- Resuspend pellet in 200 μL Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
- Lyse cells by adding 200 μL Plasmid Lysis Buffer (B2) (blue/green). Invert the tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for 5 minutes.
- Neutralize the lysate by adding 400 μL of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until the color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
- Clarify the lysate by spinning for 10 minutes at ~13000pm.
Note:. For culture volumes > 1 ml, we recommend a 5-minute spin to ensure efficient RNA removal by RNase A. Also, longer spin times will result in a more compact pellet that lower the risk of clogging the column.
- Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
- Re-insert column in the collection tube and add 200 μL of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein, and endotoxin. Centrifuge for 1 minute. Discard flow-through.
- Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
- Centrifuge for 1 minute one more time.
- Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
- Add ≥ 15 μL Hot Water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
Note: Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the DNA Elution Buffer to 50°C prior to use can improve yield.