Dh5alpha for site directed mutagenesis - (Mar/17/2008 )
I'm going to be performing site directed mutagenesis. It looks to me like stratagene's quickchange method is the easiest and most efficient. I understand too that other than the pfu enzyme - I can probably put together the reaction myself and skip the (very) expensive kit. My questions:
1. We routinely use Bioline's BioExact Long DNA polymerase in the lab. Does anyone out there know if it's suitable for mutagenesis?
2. I will be transforming into DH5alpha cells. I didn't see any DpnI inactivation in the protocol. This may be a really dumb question, but does DpnI stay out of the cells during the Heat Shock? Otherwise it would kill the cells.
from the manufacturer's website:
"The BIO-X-ACT polymerases are both an optimized blend of enzymes, containing proofreading and non-proofreading components. Due to this, a mixture of both sticky ends (with A-Overhang) and blunt ends are produced." The non-proofreading components are likely Taq which has strand displacement activity, so I wouldn't use it, also the fidelity is probably lower than pure proofreading enzyme's.
DH5alpha's can be readily transformed, for DNA to enter the cells it needs to be complexed with salts that are present in high concentration in the transformation mixture. I don't think DpnI would survive (this is just a guess). Apart from this: heath-shocking is rather inefficient (if you compare the number of DNA molecules present in the mixture and the number of cells with the number of colonies you finally get). It's plenty for molecular biology work, but it's not really efficient in the literal sense of the word. So if it would survive and kill some e. coli, you would still have colonies containing your mutant. (btw: you can heath kill the DpnI if you like).
Btw: buy components separately: you will save money!
A few years ago I had to do a similar work and found that using my own components and Pfu enzyme worked better than the kit.
As for the DpnI inactivation, it never occurred to me to think that dpnI would enter the cells .. but for me everything worked perfectly (50µl site-durected mutagenesis reaction+1µl DpnI, incubated for 1h at 37ºC, then transformed 4µl into 50µl aliquots of dh5alpha cells.)
I remember it was important to prepare the dNTP mix right before the reaction (keep dntps separately then mix then before).
As for the question abuout the enzyme you use in your lab.. well.. just try and see if it works as long as it's a high fidelity polymerase I don't see why it shouldn't work.!
After trying and succeeding I thought I'd share my experience:
I used stratagene's pfu Ultra, and NEB DpnI. I used Stratagene's online software to plan my primers. I ordered them desalted - not HPLC purified. I used regular old dNTPs at a final concentration of 250uM, 5,15 and 50 ng of template DNA and 16 cycles of PCR, 1hr Dpn1 and then Heat Shock into DH5alpha bacteria. After plating the bacteria and growing overnight I had 3-10 colonies from each reaction. the 3 I sequenced all contained my mutation.
Thanks all for the info.