PCR tips and troubleshooting

I will start this blog with PCR, not giving a protocol this time but guidelines and tips that worked for me or colleagues.

PCR seems easy at first sight, but it can turn out to be nightmarish for obscure reasons.

As a first try I will thus try to summarize my own experience of PCR.

PCR reaction usually consists in 5 steps:

a first long denaturation step at 94°C (some do it at 95 or even up to 98)

then iterative cycles of temperatures:

94/95 for 1 minute or less

50 for 1 minute or less

72/68 for n minutes (depending on size of amplicon)

and finally a step of 10 minutes at 72°C

• The first step doesn't need to be too long 2 minutes should be much more than enough.
  
• Then, the cycles can be repeated as much as 35 to 40 times, after that the "thermoresistant" polymerase should be damaged due to repeated denaturation steps, which is also why this step should not be too long (30 sec to 1 min).
  

The transition between denaturation and annealing is VERY important, despite what some could think, the slower this step occurs the more specific should be the annealing. However, it is to be noted that this transition allows for a short period of time spent at the polymerization temperature of the polymerase e.g. 72/68°C so it cannot be too long either. For tricky PCRs I went as low as 1°C/sec decrease speed, instead of 3°C/sec normally used.

• The annealing step:

The annealing temperature is also important thought it should allow for the template-complementary part of your primers to anneal to the template. For this step one should check that the forward and reverse primers are thermodynamically OK (check for concatemerization and other stuff on Netprimer, link on right hand of this page in "useful links"). Design of primers can be done through online Primer 3 service.

If the energy of a false priming or self annealing is 4 to 5 times less than the proper annealing energy then it's fine. Unfortunately I don't keep track of how bad were the ratios of my PCRs, but I will give tips on improving desperate PCRs.

Then the annealing temperature should be determined by calculation of melting temperatures of both primer, take average and remove 2°C. If they are too far from each other, just take the lowest one and remove 2°C.

• The polymerization step:
  

This step is the least difficult to set up, just look at the datasheet provided with the polymerase of your choice and it should tell you how long and at which temperature polymerization is best achieved.

• The last step is the 72°C 10 minutes. This step is in my opinion a folkloric step, that I already tried removing with success, but for the sake of traditions and also since I did it for all previous PCRs and I usually repeat them to compare results, I keep on adding it. But if you think of it this step is ridiculous since primers would not anneal properly to badly polymerized ends, so it makes no sense, in my opinion again, to add this long step at the end.
  

Optimizing your PCR:

1°) do it with different annealing temperatures and pick the best. If you don't have time for that or you don't need this PCR for precise amplification but rather to obtain a DNA for cloning, then you should try touch-down PCR (start your cycles with an annealing temp. higher than calculated and incremently decrease it at each cycle so that stringency of annealing progressively decreasses, allowing for only proper annealing in the first place so that amount of good template is largely increased when conditions are less stringent and it is then the major product of amplification).

2°) Once annealing temperature is optimized you can step up to optimizing MgCl2. Magnesium is a divalent cation that will allow negative charges of the DNA phosphate backbone to face each other when annealing. The lower this concentration is, the more specific should be the amplification of the products.

3°) You should also try to have the lowest amount of primer possible, so that primer don't dimerize due to excess, this can easily be monitored by looking at the bottom of your gel : round shaped halo, or big potato sometimes. 10 pmoles of each primer per PCR should be sufficient and I actually go for 5 pmoles in some of mine.

4°) If you need your PCR product for cloning and encounter troubles during further ligation steps, consider switching from TBE to TAE for running and buffering your agarose gels, borate is known to inhibit ligase.

5°) Think that the enzyme is provided in glycerol and it needs to be properly diluted to proceed through asmplification, too much glycerol may cause problems so take good care to dilute your reaction as advised by manufacturer.

6°) Too much DNA template may also prevent amplification, try testing series of dilutions of your template in the same PCR conditions.

7°) don't optimize too many things at a time, unless they were all independently shown to improve your PCR.

If after this you still get non specific things, than you can try hot start (adding the enzyme only at the 72°C step of the first cycle). You should also make sure that no reagent is contaminated (i.e. do a negative control without template) and if no polymerization occurs try a positive control of touch-down PCR.

When these things are checked and it still doesn't work, consider testing other polymerases and optimizing them. When things were desperate (I mean 6 or 8kb of genomic DNA to be amplified with no mistake allowed) I've used an enzyme from TaKaRa called HS LA Taq (HS: hot start, LA : long accuracy) *I'm not from TaKaRa Corp. and I usually don't like to advertise, but this brand is really worth it because not only do they sell briliantly working enzymes, but you can get sample quantities of it so you don't get charged like hell*. Some enzymes are sold with an antibody blocking them so that they only start amplifying once the antibody is destroyed after denaturation step, they are called hot start and to my experience they work better than any "melting wax" (used in the old times to prevent contact of polymerase with nucleic material before denaturation ocured) or "addition of enzyme at 72°C".

It is also possible to lower annealing temperature when too high with chemicals such as formamide. Decrease of annealing temp. due to formamide presence is best described by the following equation:

TFm = Tm - 0.61(%formamide, w/v)

Others have tried different compounds (Chakrabarti and Schutt 2001 NAR , for example)

Consider also that your template may be GC rich. I never faced that problem myself so I cannot help much with that, but polymerases are sold for these particular situations.

(see Hubé F, Reverdiau P, Iochmann S, Gruel Y. Mol Biotechnol. 2005)

Don't hesitate to post any questions or ask for advice.

Good luck !

I hope this helps