Endy:Double stranding oligo libraries
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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
- 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)
Restriction digest the library
Perform PCR cleanup on the digest
- This will remove the cut ends, since they are small.