Endy:Double stranding oligo libraries
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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 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 C for 2.5 minutes
- Cycle 5 times:
- 53 C (or whatever temperature is appropriate) for 30 s (annealing)
- 72 C for 30 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)
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
- This will either be done via electroporation or chemically compotent cells, we’re experimenting now to see which one is more efficient.
??? 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)