Endy:Double stranding oligo libraries

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Revision as of 23:34, 6 March 2006 by Kchang17 (talk | contribs) (Reaction Mix (100uL))
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Order oligos and double-stranding primers

  • Dilute stocks to 100uM
  • Dilute sequencing primers to 3.2uM (6.4uL of stock solution in 193.6uL water)
  • Dilute double-stranding primers to 10uM

Double strand the library with modified PCR

  • Total library DNA should be <25pmol per 100uL reaction
  • You want 10X the final desired amount of library by the end of PCR
    • Split into separate 100uL reactions if necessary

Reaction Mix (100uL)

Use the following reaction mix for each PCR reaction:

  • 10 μl 10x Thermo polymerase buffer
  • 10 μl 10x dNTPs (10x = 2.5 mM each dNTP)
  • 5 μl 2.5 μM FWD primer
  • 5 μl 2.5 μM REV primer
  • 1 μl Polymerase (taq or vent)
  • 66.5 μl H2O
  • 2.5 μl 10μM library stock

===PCR protocol=== ???

  • 95 C for 6 minutes (disrupt cells, separate DNA)
  • Cycle 5 times:
    • 95 C for 30 s (melting)
    • 53 C (or whatever temperature is appropriate) for 30 s (annealing)
    • 72 C for X s (elongation)
  • 72 C for 10 minutes (final elongation)
  • 4 C forever

PCR cleanup on the double-stranded libraries

  • This concentrates the samples and allows for the buffer to be switched to something more appropriate.
  • PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug)
  • Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)

Restriction digest the libraries

Separate on a gel and do a second PCR cleanup

  • Alternatively, you can run the 1st PCR cleanup out on a gel for analysis against a sample of the original library, extract it from the gel, then perform the digest. Doing a PCR cleanup on the digest will remove the cut ends, since they are small.

Ligate the sample from the PCR cleanup with a vector

Transform into compotent cells


Expected max library concentration is 10^8 molecules (this is a limit set by the transformation efficiency.) So for step 2, you would like to have 10^9 molecules for a single library transformation (more can be used so a stock can be kept.)