Lidstrom:Miniprep

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*Use 1 colony in 2 mL of TB +antibiotic grown overnight.
*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 ([[User:Janet B. Matsen|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.  
**TB gives you a higher plasmid yield than LB, but our lab currently doesn't make it ([[User:Janet B. Matsen|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.  
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* If nuclease activity is a problem, wash with [http://www.qiagen.com/Products/Accessories/Buffers/BufferPB.aspx Quiagen's PB buffer].
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** 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.
*Elute in water, not the elution buffer that comes with the kit.

Revision as of 14:14, 3 October 2012

Back to Protocols

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.

Expected Yield

  • 2 mL of pSB1A3 (high copy) should yield ~ 150 ng/uL in 30 uL.
  • 2 2mL pSB3K3 (medium copy) cultures grown in parallel then pooled should yield ~ 30 ng/uL.

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.
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