I have gel purified a specific gene from pcr, using 2% gel run at low voltage for longer time to get better separation. I used the Qiagen PCR clean up kit.
The concentration is pretty good (~50ng/ul) considering how much dna can be lost with clean up kits.
I haven’t digested the product at all, so the primer sites/ cut sites should still be intact, and the purified product shows only one band, corresponding to my control.
Can I run another PCR, if I end up wanting to get a higher concentration of the gene?
I know the salts and residual oligos can cause issues, but post-purification, is there anything I would need to look out for in order to do a standard Taq PCR?
The product from the gel extraction should be fine to use in another round of PCR. Since you're working with a single species template you will need very little input for the second PCR.
If you extended the PCR product by adding to the 5' end of your primers, you should recalculate the anneal temperature since your primers will now be completely complimentary to the template. I would also recommend checking this second PCR reaction on a gel just in case you still get non-specific products.
Sam Allsup As provided by yourself, salts and residual oligos can sometimes cause issues, but since the product is purified by gel separation, there is no much need for worrying.
I agree with John Hardy Lockhardt, but if you wish to avoid spurious mutations, use a high fidelity DNA polymerase, like Phusion or Q5, rather than standard Taq.
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