PCR with overlapping primers only (primer extention) - (Feb/26/2015 )
Hi
I need to get ~90nt fragment for ligation into plasmid. I thought the best (cheap as 90nt long oligos are already too expensive) to get this fragment would be:
ordering two oligos (forward and reverse (55 nt long)) which have 20nt overlap and then run a PCR and then purify 90nt product
As I can not find from google that people are using this approach I thought that just in case I am asking would this approach work?
I am wondering perhaps PCR like this is not very efficient?
Annealing temperature used should come from the Tm of overlap region?
Or first cycles with lower annealing temperature (according to overlap reagion), later higher Tm?
Best
This should work, but all of the action will be in the first cycle. More commonly people use Klenow fragment to do this job. Anneal, treat with Klenow + dNTPs. You can also use T4 DNA polymerase. Here's a protocol I use:
Make reactions of 10 ul
Add 1 ul each of 100 uM oligos, forward (unique) and reverse (common)
PCR cycler: 5 min/95 then 10s decreasing by 2 degrees each step to 25, then 4
Mix:
2.5ul dNTPs
2 ul 10x NEB buffer 2.1
0.5 ul T4 DNA polymerase (do not add too much enzyme, it chews back the ends)
5 ul water
Add to the annealed oligos on ice
Incubate at 12C for 20 minutes
I purify this way, but a PCR cleanup column would also work.
Purify with 40 ul Ampure, 2x 200 ul 70% ethanol wash, elution in 30 ul water
Thanks for the responce.
It seams resonable.
Just few questions:
1) Should the annealing be done in annealing buffer (10 mM Tris, pH 7.5–8.0, 50 mM NaCl, 1 mM EDTA) or NEB buffer2.1? They are about the same except BSA in buffer2.1 and EDTA in annealing buffer.
2) dNTP final concentration is 100uM as recommended in T4 DNA polymerase manual?
Best, luida
The BSA won't hurt (or you can use buffer 2 and add BSA later, or probably leave it out entirely). The EDTA is there to disable DNAse that happens to be around. But the initial heat will do that job, and you don't want the EDTA in the actual enzymatic reaction. The important thing is to have some salt to enhance annealing. I would check the NEB web site for the T4 DNA polymerase suggestons. This is what I'm currently using.