Difference between revisions of "Endy:Double stranding oligo libraries"

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===PCR protocol===
===PCR protocol===
*95 <sup>o</sup>C for 2.5 minutes
*95<sup>o</sup>C for 2.5 minutes
*Cycle 5 times:
*Cycle 5 times:
**55 <sup>o</sup>C (or whatever temperature is appropriate) for 30 seconds (annealing)  
**55<sup>o</sup>C (or whatever temperature is appropriate) for 30 seconds (annealing)  
**72 <sup>o</sup>C for 1.5 minutes (elongation)
**72<sup>o</sup>C for 1.5 minutes (elongation)
*72 <sup>o</sup>C for 10 minutes (final elongation)
*72<sup>o</sup>C for 10 minutes (final elongation)
*4 <sup>o</sup>C forever
*4<sup>o</sup>C forever
==PCR cleanup on the double-stranded libraries==
==PCR cleanup on the double-stranded libraries==

Revision as of 14:38, 28 August 2006

Order oligos and double-stranding primers

  • Dilute stocks to 100uM
  • Dilute working stocks of libraries and double-stranding primers to 10uM
  • Dilute working stocks of sequencing primers to 3.2uM (6.4uL of stock solution in 193.6uL water)
  • 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. (8bp is usually a safe bet)
    • 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

  • Expected max library size is 108 molecules (limit set by transformation efficiency.) You want to load 10X the expected library size for a single library construction. Therefore, you would like to have 109 molecules for a single transformation.
    • 1pmol corresponds to ~1011 molecules
    • Use 25pmol of library to make enough for 2500 transformations
  • Total library DNA should be less than ~25pmol per 100uL reaction

Reaction Mix (100uL, 25pmol library)

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 10 μM FWD primer
  • 5 μl 10 μM REV primer
  • 1 μl Polymerase (taq or vent)
  • 66.5 μl H2O
  • 2.5 μl 10μM library stock

PCR protocol

  • 95oC for 2.5 minutes
  • Cycle 5 times:
    • 55oC (or whatever temperature is appropriate) for 30 seconds (annealing)
    • 72oC for 1.5 minutes (elongation)
  • 72oC for 10 minutes (final elongation)
  • 4oC 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 a sample of the first PCR reaction out on a gel for analysis against a sample of the original library (double stranded should run slightly faster than single stranded), 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