Endy:Double stranding oligo libraries

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Revision as of 00:06, 7 March 2006 by Kchang17 (talk | contribs) (Order oligos and double-stranding primers)
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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
  • Some considerations:
    • Oligos should be the maximum length because this will help with PCR cleanup and ligation efficiency
    • Make sure you have some spacer sequence around the restriction site. NEB has a list of the length of the spacer sequence required for each restriction enzyme.
    • Order the lowest concentration allowable for the size oligo you want – this will be 50nmole for the 100bp oligo. This will already be more than you’ll need.
    • If you don’t mind spending more money you can order special “doped” oligo pools where instead of even concentrations of A/T or A/T/C/G or A/T/C, you get 90%A/2%C/8%G, etc. This allows for you to generate a library which is much more likely to produce productive clones.

Double strand the library with modified PCR

  • Total library DNA should be <25pmol per 100uL reaction
  • You want to start with 10X the final desired amount of library for 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.) We will typically want a library to have approximately 10^10 molecules (~0.1pmol)