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 &mu;l 10x Thermo polymerase buffer
 * 10 &mu;l 10x dNTPs (10x = 2.5 mM each dNTP)
 * 5 &mu;l 10 &mu;M FWD primer
 * 5 &mu;l 10 &mu;M REV primer
 * 1 &mu;l Polymerase (taq or vent)
 * 66.5 &mu;l H2O
 * 2.5 &mu;l 10&mu;M 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

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) [[Image:Double-stranded_oligo_libraries.jpg|thumb|none|300px|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.