Endy:Double stranding oligo libraries

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)

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.

Transform into compotent cells

• This will either be done via electroporation or chemically compotent cells, we’re experimenting now to see which one is more efficient.

Notes

??? 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)