Here is my workflow for high plasmid yields from cultures of DH5α E. coli. I use the GeneJet Plasmid Miniprep kit from Thermo (#K0502), but most spin column kits work the same. Generally I have to extract plasmids from numerous cultures simultaneously, so this protocol also gives guidance on the timing of each step.
- Prick a colony of E. coli with a sterile toothpick or a 10 µL pipet tip or add a loop from a glycerol stock.
- Inoculate 5 mL LB plus the appropriate antibiotic in a 25 mm culture tube or 50 mL conical tube.
- Incubate overnight (12–16 hours) at 37 ˚C with shaking at around 250 rpm.
- The next day, warm up a block heater to 65 ˚C and preheat Elution Buffer (10 mM Tris pH 7.5–8.5).
- Decant the entire culture into a 15 mL conical centrifuge tube. If you have numerous samples, start with 8–10 cultures here.
- Centrifuge at 4000 xg for 5 min. Some bacteria may still be suspended, but that is okay as long as there is a nice pellet.
- Decant LB from 4–5 tubes and place them upside down on a paper towel to remove residual LB.
- Add 250 µL Resuspension Buffer with RNase A added (This buffer is stored in the refrigerator).
- Resuspend by pipetting up and down vigorously.
- Add exactly 250 µL of suspension to a labeled 2 mL microcentrifuge tube.
- Add 250 µL Lysis Buffer, invert the tube 3–4 times, and incubate at 65 ˚C for 5 min. Do not incubate much more than 5 min (Note 1).
- After lysing for 5 min, add 350 µL Neutralization Buffer to one tube at a time and immediately mix by inverting gently 5 times. It should make a white chunky precipitate (Note 2).
- Centrifuge at 12,000 xg for 5 min (Note 3).
- Decant supernatant into a labeled spin column. Cap the column and invert twice (Note 4).
- Centrifuge for 15 s at 12,000 xg and discard flowthrough.
- Add 500 µL Wash Buffer (Note 5) and centrifuge for 15 s at 12,000 xg. Discard flowthrough.
- Repeat step 7 for two washes. At this point you may keep the columns at 4 ˚C until all samples are completed.
- Centrifuge empty columns for 2 min at 12,000 xg to dry the columns and remove residual ethanol.
- Place columns into new labeled 1.5 mL microcentrifuge tubes.
- Add 40–50 µL warm (65 ˚C) Elution Buffer (10 mM Tris pH 7.5–8.5) directly onto the center of the silica column and incubate for 2 min at room temperature. If you miss the silica column, the DNA will not elute properly.
- Centrifuge for 2 min at 12,000 xg
- Quantify your plasmid DNA. I typically yield 200 ng/µL on average with some samples yielding up to 1150 ng/µL.
Note 1: While these four are lysing, repeat Steps 7–11 with the next 4-5 cultures. These steps take me about 5 min to complete depending on how stuck the pellet is. If you have finished your first batch, then you may start Step 5 and Step 6 with additional 8–10 cultures.
Note 2: If you have multiple batches of samples, you can pause at this step and start with Steps 7–11 for the next 8–10 samples. It is okay to keep them in this buffer for a small amount of time. I have not seen degradation within 30 min.
Note 3: Make sure the lid opening is facing the center, so that decanting the supernatant becomes easier later.
Note 4: Make sure there are no precipitates introduced here. If some does get stuck, remove with a 1000 µL pipet tip.
Note 5: Ethanol from the wash buffer evaporates over time. At the end of the day, mark a line on the bottle to indicate the buffer level. Close the lid very tightly. If some evaporates, fill to the level line with ethanol. Alternatively, Wash Buffer is easy to make from scratch. I found that 65% ethanol in 10 mM Tris (pH 7.5–8.5) is sufficient to wash the plasmids.