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

Hi, I have been attempting overlap PCR with  770 bp and 4500 bp fragments. They have a 26 bp overlap region. Both fragments were gel purified prior to overlap PCR. Here is the setup.

 

Q5 10X buffer: 10ul

Primer F: 2.5ul (10uM stock)

Primer R: 2.5ul (10uM stock)

GC Enhancer: 10ul

dNTPs: 1ul (10mM stock)

PCR fragment 770bp: 1uL (150ng)

PCR fragment 4.5kb: 1uL (100ng)

Q5 high fidelity polymerase: 1uL

dH2O to 50uL

 

PCR cycles

 

98C - 30s

50C - 60s            x35

72C- 6 min.

 

I attached the gel image. Lanes 3 and 4 show the correct band (among others) as indicated by the arrow, but it so faint that I can not move forward since i must still double digest it. Any advice on how to optimize this would be appreciated. It seems that the smear is the big issue here as well as the strong band at 600bp. The middle two bands I assume are my template

The two fragments - have you annealed them?  I'm guessing so, given that you actually got a band, but it pays to check.

 

A 5 kbp PCR isn't going to be all that easy to get going well, independent of the overlap issue.  Is there any chance you could run some temp gradients for the primers?  You could also try some sort of touchdown PCR.

I actually dont anneal them prior to PCR. I guess I just use the first cycle to anneal them. I have had success in the past with overlap pcr with my methods, but you may be right that the 5kb fragment is the challenge. previously i was working with 2-3kb fragments.

 

What I have tried today per NEB FAQ's is using 1. smaller amount of polymerase 2. 65C for extension and 3. raise the annealing to 55C. I will determine if this improves the yield, and then I may move to touchdown PCR. I just use touchdown PCR as a last resort because in my 3-4 years doing my research I have never had touchdown PCR improve my PCR. Usually my touchdown PCR gel looks identical to conventional PCR.

 

ALSO, I gel purified the faint correct size band and used it as a template. That is lane 2 of the gel I posted.

I'd have a go at annealing the two fragments before the PCR, just in case.  If you have them nicely purified, just follow my attached protocol.

 

 

Thanks for the protocol. I will give it a try and see how it turns out. How stringent is the buffer requirement? Can I just use my PCR reaction buffer to mix the fragments for annealing?

You can use a big range of concentrations of the DNA, but I think the salt content of the buffer is pretty important.  I would guess that PCR buffer would work, as it is normally used for PCR which has a lot of annealing going on.

I see something what may cause a problem in your protocol.

 

You wrote you are using 150 ng of 77bp product and 100 ng of 4.5kb product.

 

That is similar concentration. But both products need to have very similar molar ratio to actually maximize the annealing. The number of molecules count.

 

150 ng of 770bp product means 1.8 x 10¹¹ molecules.

 

100 ng of 4.5kb product means 2 x 10¹⁰ molecules.

 

That is 10 times more of the small product.

 

Also, though I have been overlaping only small fragments, my hotstart protocol includes 15 minutes of 95 deg denaturation prior to cycling. I too didn't do any separate annealing, but I think denaturation and the right mix-up of both fragments is more important and more difficult in such big fragment/combination. I would definitelly consider more care about this step.

Thanks for the input, I repeated with similar molar ratios and the band remained faint with a smear. The higher annealing temperature along with 65C for extension has improved the yield slightly, but still may not be sufficient to move forward with restriction digests. Im only getting around 100ng per 50uL PCR reaction. By the time I purify, digest, and purify, I likely wont have anything left. I am going to switch enzyme (Pfu high fidelity instead of NEB Q5) to see if this will make a difference.

 

Im looking into Gibson assembly. On paper, it seems like it is a grand slam, has anyone had success with the Gibson assembly from NEB?

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.

(15 min/98 deg was not a recommended step, I just used it as an example that during my reaction, the denaturation is by design longer, since my enzyme requires activation such long every time)