In addition to what Jesper Nielsen asked, what results have you gotten with your troubleshooting?

If you are getting smears that include the size of your target amplicon, a nested PCR will greatly increase your specificity.

If you are getting zero bands at all and only primer dimers, then Bernd Zorr's suggestion is spot-on, because if only 25% of your primer is binding to your target, your TM is effectively only those complementary basepairs, in addition to a penalty due to your mismatches. In this case, lower TM and increase MgCl or S04 concentration depending on the polymerase you are using.

If you are getting a really bad smear, an alternative avoiding nested pcr is doing a touchdown program: the first cycle has an extremely high annealing temp, ~72 for most of your polymerases. Every subsequent cycle, you decrease it by .5-1 degree until you hit 50-55C (this temperature can be optimized according to your primers, for this I would suggest 55). Once the cycle is at 55C annealing temperature, you do an additional 20-25 cycles at the low temperature.

Touchdown exploits the fact that while binding is low at high annealing temperatures, it is highly specific, so the only products that will be amplified during that time is your product of interest. As you lower the temperature, you increase binding, but your primary product already has a head start and immediately outcompletes any off-target amplicons exponentially. I use touchdowns for very tricky pcrs off of full genomic dna where complexity is high and your target is low in enrichment.

Finally, if it is true that your primers are only 25% complementary, what is stopping you from having full complementarity? Or is the 75% actually an overhang that you wish to add onto the ends? If so, a two step program as suggested by Bernd Zorr would be spot on. You do 5-10 cycles at the Tm of the initial complentarity, and then bump up the annealing temperature to the Tm of the full primer binding. I regularly do long-range (5-8 kb) pcrs with 25-50 bp overhangs using this method.

Good luck!

Hi Deepak

Please explain what you mean by 25% complementary. Do you mean that the oligos are only partially complementary over their entire length or are the 3' ends fully complementary?

Jesper

In addition to what Jesper Nielsen asked, what results have you gotten with your troubleshooting?

If you are getting smears that include the size of your target amplicon, a nested PCR will greatly increase your specificity.

If you are getting zero bands at all and only primer dimers, then Bernd Zorr's suggestion is spot-on, because if only 25% of your primer is binding to your target, your TM is effectively only those complementary basepairs, in addition to a penalty due to your mismatches. In this case, lower TM and increase MgCl or S04 concentration depending on the polymerase you are using.

If you are getting a really bad smear, an alternative avoiding nested pcr is doing a touchdown program: the first cycle has an extremely high annealing temp, ~72 for most of your polymerases. Every subsequent cycle, you decrease it by .5-1 degree until you hit 50-55C (this temperature can be optimized according to your primers, for this I would suggest 55). Once the cycle is at 55C annealing temperature, you do an additional 20-25 cycles at the low temperature.

Touchdown exploits the fact that while binding is low at high annealing temperatures, it is highly specific, so the only products that will be amplified during that time is your product of interest. As you lower the temperature, you increase binding, but your primary product already has a head start and immediately outcompletes any off-target amplicons exponentially. I use touchdowns for very tricky pcrs off of full genomic dna where complexity is high and your target is low in enrichment.

Finally, if it is true that your primers are only 25% complementary, what is stopping you from having full complementarity? Or is the 75% actually an overhang that you wish to add onto the ends? If so, a two step program as suggested by Bernd Zorr would be spot on. You do 5-10 cycles at the Tm of the initial complentarity, and then bump up the annealing temperature to the Tm of the full primer binding. I regularly do long-range (5-8 kb) pcrs with 25-50 bp overhangs using this method.

Good luck!

Thanks for the answers, guys. I guess some more clarity is needed in my question. So, here goes.

So, I have two primer sets. The primers are long and 19-21 bp at the 3' end of each primer are complementary to the target (in this case, a plasmid template with my gene of interest). The remaining ~75% of the primers have restriction sites and tags for subsequent steps of cloning. I expect amplicons of 0.4 and 0.6 kb from the two primer sets.

The results of the troubleshooting PCRs were depressing (needless to say!). I did not get any amplification with Q5, Phusion, GoTaq or in the gradient (with GoTaq). With the TAIL PCR (with GoTaq), I got a faint smear but they were lower than the expected size, with both primer sets.

After I posted the question, I've done both step-up and step-down PCRs (5 cycles at temp1 followed by 25 cycles at temp 2, in both step-up and -down). I used Tms much lower (50˚C and 58˚C) than the primer Tms. Like I saw with the TAIL PCR, I see a faint smear, but again of a lower than expected size.

What I haven't tried yet is a touchdown PCR (thanks Brian). That is on the cards now.

Any suggestions with this additional information, guys?

Thanks,

D

95˚C

How big is your amplicon? And is the template you are using complex?

Assuming your amplicon is large, since you shouldn't have this issue with small fragments, the quality of your template may be responsible for the smears, considering how much troubleshooting you have done already.

Follow up questions to that are: have you assessed quality of the template by nanodrop/agarose gel? Shoulders on either end would suggest contaminants. If by gel, you don't get a clean single band where your template should be, or a compact smear if you are doing genomic, consider gel extraction to remove extraneous sequence.

If it is genomic, potentially there are BAC clones of the region you are looking for? That greatly simplifies your PCR.

Finally, what is the GC content of the amplicon? >55% and you might want to consider adding in ethylene glycol, 1,2 propanediol, betaine, DMSO, or DMF, in that order of preference. In fact, if you at any time get a super faint band that is the size of your target, consider adding those, they have aided me in PCRing large fragments otherwise impossible.

Also, I hope your PCR machine allows decremental changes in cycle temperature, we only recently got one that could, previously touchdown pcr was 10 minutes of typing in each single step! :p

Good luck

The amplicons are small, only about 500 bp and I still have this problem! I have checked my plasmid template and since getting these issues, am using gel purified plasmid. I've checked and double checked my primers to ensure that they are in fact complementary to the template.

I've also tried with and without DMSO, even though the template is not GC rich, to no effect.

I'm running a touchdown PCR now. Will have the results in a couple of hours. Fingers crossed!