Overlap PCR, need help - (Dec/05/2013 )

phage434 on Sat Dec 7 04:28:43 2013 said:

Two things:

1) That is MASSIVELY too much DNA for a pcr template. Almost certainly that is the major problem. Try again with equimolar amounts down more at the 1 ng level, or even well below that.

 

2) Why are you adding 10 ul of a 10x buffer, and then bringing the volume to only 50 ul. Also, that sounds like too much of the "enhancer".

 

I don't think you should be doing 15 minutes at 98 -- unnecessary and likely damaging the enzyme and especially the dNTPs.

 

I think you can cycle this as:

 

98/2 min

 

30 cycles:

98/15s

50/10s

72/3 min

 

72/5 min

 

Q5 is a very fast enzyme -- 2 kbp in about 30 s. You can probably do the extension at 1:30 min but why press your luck.

 

50 seems a bit low for an annealing temperature -- is this a result of Tm calculations? I would normally start optimizing at 55.

Thanks for these tips. I will reduce the template to around 1ng of PCR product. I have successfully amplified small amounts of my overlap product at 55C, so I will continue with this. I just typed the conditions wrong, I have been using the right amount of buffer and enhancer (5X). The reason I have been using so much template is the last time I did overlap I used these amounts and it worked wonderfully. Apparently, I was just getting very lucky.

RESOLUTION:

I attempted the overlap PCR with 2ng of each template, but I couldnt get a product. I went back to my original conditions that gave a smear, but a faint band and cut the bands out and combined them prior to restriction digestion. After ligation, I got a correct clone, so I can move forward. I guess you don't always have to optimize the PCR :)