Howto: Maximum possible yield from a PCR? - What to optimize: Mgcl/primers/Polymerase/enhancers? (May/25/2007 )
I need around 5-10 ug of DNA from a PCR for electroporating eggs. I don't even know if that is feasible by PCR.
If you think that it is:
- How could I possible achieve this? I have tried three different quantities of: MgCL - temperature - primer - NTP - template. my current maximum yield is around 1ug /50ul@ 35 cycles with a standard Taq.
- Do you think that a better Enzyme (Phusion?) could help?
- Or shall I rather add enhancers like betaine?
- Or try continue improving the conditions? (noteable increasing Mgcl)
Or shall I rather stop trying getting as much as 5-10 ug of DNA from a PCR?
Thanks a lot!
1ug from PCR is a decent yield.
If you want 5-10 ug, start 5-10 PCR reactions and pool them for 5-10 ug of DNA.
How about doing ten 50ul PCR vials, since you already have the PCR conditions sorted.
10 vial will give in total 10ug according to your calculations. 1ug is not a bad yeild.
But if you want to monkey around the polymerase, you need to consider how long is your PCR product? If it is short, less then 1kb, Taq is your best bet. For something small, it is the highest yeild and cheapest option. Of course there are many types of mutant taq available. I do not know which is the best.
For something around the size of 2kb to 5kb I would recommend Kod Hifi, it is relatively "cheap" and produces good yeilds. Phusion is only good for amplifying very long products >5kb, for the relative high cost and lower yeilds to be justified.
Increasing the acount of Mg ions will certainly increase the yeilds, although it comes with the cost of increased none specific band formation.
Other components like BSA, glycerol, DMSO, betaine can be added. But I don't believe they will make a large difference to your yeilds as they are already good.
If I were you, I will use ready to use hotstartTaq PCR kit
click on the link to read about it
It is fantastic every thing is ready to use, all what you need is to add only your DNA and primers on the master mix. It will give you sharp bands without optimization no ice and the preparation will not take few minutes.
I think there are some other companies but I tried this one and I did not need to repeat my PCR
Hi and thanks to everyone for the feedback.
OK, if I cannot get 5 ug yield from a reasonable amount of PCR vials, I rather try to amplify in bacteria then. Yes, I know that I can pool PCRs but in this case I guess I'm simply demanding too much from my PCR.
And I don't think that a hotstart enzyme will increase the yield a lot, thanks for the suggestion.
I'm now playing around with PCR-induced cloning (double fusion pcr + hybridization), without ligation, where I can generate plasmids in the PCR tube and then directly transform bacteria afterwards. It might take a little more time, but I would give me 100 times more DNA and does probably make more sense in the end for my experiment. I'll post my results to the forum (especially when I run into trouble... :-) when I know more...