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Tips You (Mostly) Won't Find in the Manual
- Use 1 colony in 2 mL of TB +antibiotic grown overnight.
- TB gives you a higher plasmid yield than LB, but our lab currently doesn't make it (Janet 2/23/12). If your plasmid is high-copy number, one tube is sufficient. If you have a medium copy plasmid, grow 2-3 cultures from the same colony in parallel and pool them before loading the column.
- If nuclease activity is a problem, wash with Quiagen's PB buffer.
- Buffer PB contains a high concentration of guanidine hydrochloride and isopropanol. The exact composition of Buffer PB is confidential. However, this buffer can be purchased separately.
- Elute in water, not the elution buffer that comes with the kit.
- The elution buffer stabilizes DNA and reduces DNase activity, but comes at a cost -- the added chemicals are likely to lessen DNA sequencing and subsequent DNA manipulation successes.
- The amount of time the overnight culture grows is quite flexible.
- There is literature that suggests what stage in growth is optimal to harvest plasmids. People usually just do whatever is convenient.
- 2 mL of pSB1A3 (high copy) should yield ~ 150 ng/uL in 30 uL.
- 4 mL pSB3K3 (medium copy) culture should yield ~ 30 ng/uL in 30 uL
- Make sure the air:liquid ratio is at least 5:1
- Our lab prefers the Fermentas/Thermo Scientific miniprep kit over Quiagen because itis cheaper. -Janet 10/2012
- Do not grow cultures with less than 4:1 air:liquid ratio -- it is very bad for plasmid prep. This is still one of Janet's open questions 2/23/12
- You can elute in 30uL instead of 50uL to get a more concentrated sample.
- Record the A260/A280 measurement down when you record the concentration. It should be between 1.8 and 2.
- You can leave the cap on the fresh eppendorf tube you elute in. This allows you to pre-label the tube and stick it straight into the freezer after you are done instead of pipetting each into a fresh tube.