Endy:Double stranding oligo libraries

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

• 95^oC for 2.5 minutes
• Cycle 5 times:
  • 55^oC (or whatever temperature is appropriate) for 30 seconds (annealing)
  • 72^oC for 1.5 minutes (elongation)
• 72^oC for 10 minutes (final elongation)
• 4^oC forever

Perform PCR cleanup on the double-stranded library

• 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, so multiple 100-ul reactions of 25pmol can be combined into one column
• Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
• You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)

  

  Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)

Perform PCR cleanup on the digest

• This 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.