Quick cloning question - Purification of PCR fragment (Sep/02/2005 )
Hi,
I'm cloning a PCR fragment with primers designed to introduce restriction sites. I digested off the sequence upstream of the restriction site following PCR and now I have a question. Should I PCI/ethanol precipitate my fragment from the restriction digest mixture? I have a protocol here that seems a bit redundant... it has me PCI/EtOH precipitating my fragment and then gel purifying. Gel purification alone should weed out my insert from the digest components, right?
I just thought I'd ask. I'm an undergrad and am supposed to religiously follow protocols that are given to me, but this seems a little strange. It seems as though either method on it's own would yield the purified fragment that I want to ligate (though I know I'd lose a bunch with gel purification).
Thanks,
Hank
I'm cloning a PCR fragment with primers designed to introduce restriction sites. I digested off the sequence upstream of the restriction site following PCR and now I have a question. Should I PCI/ethanol precipitate my fragment from the restriction digest mixture? I have a protocol here that seems a bit redundant... it has me PCI/EtOH precipitating my fragment and then gel purifying. Gel purification alone should weed out my insert from the digest components, right?
I just thought I'd ask. I'm an undergrad and am supposed to religiously follow protocols that are given to me, but this seems a little strange. It seems as though either method on it's own would yield the purified fragment that I want to ligate (though I know I'd lose a bunch with gel purification).
Thanks,
Hank
Ya, the PCI/etoh step does seem a little redundant. What I do is first run a gel with 3 - 5ul of the PCR product (not restricted yet) to check for obvious non-specific products and whether the PCR has worked. If all is Ok (i.e. a single band of the expected size is present), then I gel extract the band (from a much larger PCR product volume, say abt 150ul).
Yes, gel extraction is quite wasteful on PCR product but this reduces greatly the likelihood that u will get other non-specific products. Then I restrict the gel-extracted PCR product.