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I am trying to do a PCR using a plasmid as the template, pretty basic stuff. But the problem is that my forward primer just happens to bind to another location on the plasmid in the reverse orientation, in between the proper forward binding site and the reverse binding site. This isn't an example of non-specific binding, the sequences are perfect because the plasmid contains self-complimentary regions and my forward primer just happens to land right on it.

Here is a diagram:

enter image description here

Simply optimizing the melting point or magnesium concentrations probably won't help, because the sequences are the same. I'm doing this cloning to insert a reporter gene into the vector, and I need to place it very precisely, so I don't know if I can order a new set of primers, the insert needs to fit there.

The bad PCR product is about 500 bases shorter than the good product, but I only see the bad band on gel when I run a normal PCR. If I reduce the amount of forward primer and increase the reverse oligo, I can see the good band, but the bad band is still there and the total yield is much lower.

So my idea right now is to do an asymmetric PCR using the reverse primer only, then add forward primer to create the double-stranded DNA, then separate the good and bad bands using gel electrophoresis. But I don't have a clue how many cycles I should run each step, or how much forward primer to add, etc. Additionally, asymmetric PCR has low yield due to arithmetic amplification, but once I add the forward primers it can begin symmetric PCR and geometrically amplify both bands, but I think the shorter bad band wins out.

Does anyone here have experience with this sort of complex PCR, or suggestions to improve my yield?

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    $\begingroup$ What's the problem wtih using a lower percentage gel and running it for a long time on low voltage to separate the two bands and cut out only the longer one you want? $\endgroup$ – Armatus Apr 9 '18 at 11:07
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    $\begingroup$ Can you amplify two separate regions that meet at a restriction site and reconnect them by ligation? Without knowing the exact sequences and purpose, I'm afraid it's hard to give clear recommendations, because the best way to approach this kind of thing tends to be highly specific. $\endgroup$ – Jack Aidley Apr 9 '18 at 14:51
  • $\begingroup$ @Armatus When doing the PCR using equal amounts of both primers, I did not see the desired 3000 base band, only the bad 2500 base band on gel. The ladder had bands at both 3000 and 2500, so I know the bands should have resolved if they were both present in the PCR sample. The shorter band must be winning out. I was able to see both bands when I used much more reverse primer than forward primer, but the bands were very faint. $\endgroup$ – user137 Apr 9 '18 at 15:02
  • $\begingroup$ @JackAidley I designed the cloning procedure around InFusion cloning, but that system can work with multiple fragments as long as they have a 15 base overlap. I can probably design a couple more oligos that bind on top of a restriction site. That will let me cut the plasmid and PCR the shorter pieces with overlapping ends in separate reactions, then mix all the parts together for the InFusion reaction. I'll let you know how it works, but it will take time to get the new oligos. $\endgroup$ – user137 Apr 9 '18 at 15:06
  • $\begingroup$ @user137: if you combine the fragments by PCR won't you encounter the exact same problem with your fusion (aka stitch) PCR? $\endgroup$ – Jack Aidley Apr 9 '18 at 15:14
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From experience, your present problem will not completely go away no matter what you do. The idea for asymmetric PCR is not a bad idea. However, you will only get linear amplification of the desired product from the initial single primer, so you will not "see" any product even after 30-40 cycles. This first single primer PCR might also cause downstream problems with amplification of undesired products made during the second PCR (junk products).

I agree with comment #1, but then neatly cut the "visible" "good" band out of the gel. Suspend (crush) the agarose band in sterile TE buffer, and then use an aliquot of that as template for a secondary PCR (use equal primers - maybe 30 cycles). This should provide more than enough "good" starting template.

Alternately, you could design a new forward primer that does not have a bad secondary location on your plasmid.

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