Lidstrom:Miniprep

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Tips You (Mostly) Won't Find in the Manual

• 2 mL of TB +antibiotic grown overnight yields good minipreps
  • TB can be used instead of LB
  • Warning: cells get growing in LB faster than TB. If you have small colonies you are inoculating from, the colonies may not be ready the next day. Only use TB if you are adding a large colony as the inoculum. --JM 6/2015
  • TB gives you a higher plasmid yield than LB, but TB requires a little more prep work than LB because you have to add filter sterilized phosphate solution after autoclaving the rich base. 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.

Expected Yield

• 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

Miscelaneous

• 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.