I found an interesting (old) paper about long range PCR: W. Barnes et al. PNAS 1994. In there they note about this phenomenon as well "especially at high enzyme levels".

In a small pilot experiment I tested this, and it seems right. (see attachment): when adding 1ul extra PfuUltra II fusion HS [Agilent] in 50ul, plasmidcontrol 0.001pg is suddenly inhibited and material got stuck in the well. [dT-traag sample is HIV-cDNA, added in 2 different quantities]

Furthermore this paper is interesting in explaining that high temperatures should be avoided/minimized for long templates. For my 9kb fragment, only 2secs denaturation at 90C works well. Some further optimization needs to be done, but when the ratio between enzyme-amount and template is out of balance, signal seems to retain in the well.

I can't see your attachment??

Could it just be your template DNA? Possibly your PCR isn't working and you have too much template DNA that's visible in the well?

9kb is a long fragment that may benefit from using a long-range specific polymerase.

I diluted the DNA from 10pg until 0.0001pg. You don't see that on gel. I will check for the attachmentfile (?)

thanks

uploading with Google Chrome instead of Internet Explorer does the trick!

How many picograms did you use per lane then? EtBr has a detection limit in ng range, Sybr green (as example) 30-60 pg. Don't know your DNA concentration, but if you have 0.0001pg/microL, then you'd need more than 100 mL sample to see something (if I calculated right, it was a quick and dirty calc).

And what about the agarose concentration? The lower the better with such large fragments (i.e. 0.7-0.8 %).

Ah, Its definitely a PCR product then!

I see your attachment now!

I was going to suggest that it may be polymerase stuck to your amplicon, but I think you have tried adding SDS to your loading buffer? You could try cleaning the product before running on the gel anyway to see if it helps.

@P.Hondelmann: gel is 0.8% The DNA amounts are about the plasmid I put into the pcr. It doesn't seem to be a sensitivity problem, but more that it sticks into the well.

Sometimes I do get the correct band of 9kb into the gel under the same conditions?!

What about purifying your PCR product? I agree with Amanda's suggestion that the polymerase might be stuck to your amplicon.

I had a similar Gel image when I tried to run a Gibson Assembly reaction. in the mix I had a 6kb plasmid and three enzymes (exonuclease, ligase and polymerase). That stopped everything from running into the gel (confirmed by the technical assistance from NEB).

I can definitely try to purify the pcr product first before loading, altough in some pcr's I ran under same conditions did not give retention in the gel.

I've seen very AT-rich regions of DNA stick to DNA-modifying enzymes. Parts of the HIV genome may fall into this category, giving more reason to try to purify this fragment before loading.

Several people have reported seeing this phenomenon, with different outcomes altogether. There is currently limited information about your process to offer any specific advice.

But...

Looks like your PCR simply didn't work. What you are seeing on the gel appears to be some sort of contamination (high-molecular weight smear). However, I think it may be something much simpler than concatemers.

Start with fresh stocks (primers, dNTPs, buffers, polymerase, water, etc...) and retry. Also, use a different batch of tubes, as it appears you are seeing this smear in all your lanes, regardless of the DNA concentration used. I don't think you are overloading your PCR reactions at this time (in fact, increasing it a bit could help you, see 10pg lane). I am not sure if this is gDNA or plasmid DNA, but you may not have enough templates for amplifying such a large fragment.

I am not sure what polymerase you are using, but some polymerases (such as Phusion) do not play well with tweaking MgCl2 concentrations. Start simple, check your primers for degradation, and use a new batch of PCR tubes.

Good luck!

my dear friend,

can you tell me which polymerase you are using.

I use PfuUltra II Fusion polymerase of Agilent.

I attached more info about conditions plus extra photo

Hi P.Boers, I have the same unsolved problem. One suggestion a "PCR expert" gave was to cleave the PCR product randomly, using a restriction enzyme, I haven´t try it because I don't have any Ez available right now. I am working over a cDNA synthesized using three priming strategies (Rad6-mer, oligo(dT) and Gene Specific primer). Once I run the PCR in 0.8% agarose gel with those samples I see a condensed gelatinous mass in the PCR product I also include a gDNA in parallel as a positive control sample and I don´t see this effect on it, neither on the NTC, just on the cDNA samples. Please if you get any solution, share it with me, I will try other suggestion such as adding BSA that theoretically will chelate some aggregative bodies in the reaction that could be agglutinating the PCR products. ASAP I can solve the problem I will post the solution too.

thanks Ariel. I will do so...

In the end I need this pcr for cDNA as well. Good luck

Wondering if you had any luck solving this?  I am having a similar issue with amplification products from my long distance PCR (after cDNA synthesis using viral RNA)

@ E.Rams:

There is no clear answer yet, unfortunately. The big question is whether the signal in the wells is specific product at all? When there is a lot of specific PCR product, there is no/hardly any retention in the well. I focussed on aliquotting primerstocks (less freeze/thawing) and use of low-DNA bind plastics (primer aliquots, +contr plasmid-dilutions and 0.2ml PCR tubes). At least that gave me more consistent positive results.

Thanks for the reply... I tried some of the other suggestions (SDS in loading buffer, increased voltage and lower percentage gels) and am thinking it is a non-specific product of some sort also.  I will also keep your other suggestions in mind.  Thanks again

Hi Boers, I also have the same problem now, you can see in the attachment my pcr product stuck in the well only very minor portion of it got resolved(left well). I tried in low % gel( i e 0.8% agarose gel) and also used PCR purification kit followed by gel extraction but did not work out for me. if you have any suggestions i will be happy for that.

I am also having the same problem.

I found an interesting (old) paper about long range PCR: W. Barnes et al. PNAS 1994. In there they note about this phenomenon as well "especially at high enzyme levels".

In a small pilot experiment I tested this, and it seems right. (see attachment): when adding 1ul extra PfuUltra II fusion HS [Agilent] in 50ul, plasmidcontrol 0.001pg is suddenly inhibited and material got stuck in the well. [dT-traag sample is HIV-cDNA, added in 2 different quantities]

Furthermore this paper is interesting in explaining that high temperatures should be avoided/minimized for long templates. For my 9kb fragment, only 2secs denaturation at 90C works well. Some further optimization needs to be done, but when the ratio between enzyme-amount and template is out of balance, signal seems to retain in the well.

have you solved the problem? I am having the same problem

In case of long PCR, you need to decrease the concentration of the starting DNA (particularly if you are amplifying a whole plasmid), because their is high risk of self priming

Hey mates, I'm facing the same problem. I think when you have not enough pcr product, the ratio of protein to DNA is higher and this cause the sample stuck in wells. I'm trying to make site directed mutagenesis in a plasmid. As you see in the attached file those reactions have pcr products show less amount of sample remained in wells.... I'm going to augment the extention time, probably this would show the accuracy of idea...

Hey everyone, I increased the elongation time, still the result is the same. PCR product stucks in the wells. Next I'm going to change DNA concentration to see if self priming is the problem or not? I'll update you...

Hi all,

I had the same problem before that was solved by reducing both extension and annealing time and increasing annealing temperature by 2-3 degrees to omit any interference from non-specific binding. you should also avoid long denaturation time to prevent DNA damage.