Hi, I have a problem with not working PCR. I am trying to amply two fragments next to each other on chromosome with Q5 high fidelity polymerase. I had problem with amplification of both. I tried dilute new primers from stock, change annealing temperature based on NEB calculator, gradient PCR, changed time of annelation, changed dNTP, concentration of template. Finally I excluded enhancer and I amplified one fragment. The other is still not working. Not even primer dimers. I also checked if i designed primers in a right way. And everything is ok. Any help? Thank you.
Hi Andrej Minich
If absolutely nothing is being amplified, not even off-target DNA, it is very very strange (as you said). If you haven't done it yet, I would do the following:
- I imagine that your cDNA is in good condition and does not have some type of contamination that could interfere with the activity of the polymerase, you know... And that you use a proper thermal cycling profile
- I would check the mix you use: proper MgCl2 concentration, enhancers (it is so weird that you have better results when removing the enhancer), dNTPs concentration,....
- Check your primers again and amplicon size (What software did you use to design them?)
- Annealing temperature must be calculated empirically by using a thermal gradient, not based in "in silico" software (can guide you, but only that, you know...)
If you could give us more details or images of the amplification curve and melting curve, we could narrow down where the fault is, if the DNA and the reagents, primers,... are correct.
Hope you get it. Good luck
Angel del Marco everything is ok with my template. Usually to amplify anything, i work with lysate. Since I have got no results, i isolated chromosome and now i am working with that.
In a case of MgCl2, the buffer for Q5 polymerase contains MgCl2 already, so I am not sure i can work with concentrations.
In a case of enhancer, my primers are quite long, mostly As and Ts and enhancer is good for GC rich primers and regions. That is the reason that i excluded enhancer. And it worked. Even for my fusion PCR.
I already checked my primers and amplicons. I used snapgene. And everytime i check online primers do not create duplex and so on. But always, when i work with new primers i use thermal gradient pcr to find optimal temperature.
All in together, I used this "protocol" for many of my amplifications and i got at least non specific amplicons but also mine desired. First time in this case, I have no amplified fragments at all.
Additionaly, even for pcr that worked one day, next day with the same protocol, i have no results.
Do you want to amplify both regions at the same time in one reaction? If not, the easy fix is to set up two separate PCR reactions (one for each region of interest). It's not always possible to multi-plex reactions. You will likely have to test out several pairs of primers for each region of interest.
If your regions are REALLY close together (less than 10,000 bp), you can amplify across both with one set of primers. But it would mean a really long reaction time and pricey enzyme.
Good luck!
Katie A Burnette Hi. I work with two separate reaction mixes. For each fragment. And each fragment is around 1000 bp long (fragments for homologous recombination).
I even tried to do multiplex PCR with all primers together, to get at least non specific amplicons. But again nothing.
Thank you for your advice!
Primer stocks can degrade over time. Try ordering a fresh set & see if that helps.
Hmmm, that is odd. Ask someone else in your lab for a set of primers & DNA that they know gives a bright product. Set up the PCR at your work station & thermocycler & the high fidelity taq. If that doesn't work, then the issue is not unique to your primers & gene of interest. If you do see a band, then you know the issue is unique to your reagents (or thermocycler setting).
It might be an issue with the DNA polymerase mix, the thermocycler, the amount of DNA you add, etc.
Time to start ruling out the basics.
Katie A Burnette thank you for your advice. But i already tried 2 other thermocyclers and also changed my work station. I have not tried other polymerases yet but my coworkers use the one from microtube i was using and they have results.
i think, the problem is with primers. Because i ordered mine with other colleague and he has really high annealing temperature (68 celsius). And the priemrs were designed for 55. And also has very weak band (even trying gradient). so i think something went wrong with whole order of primers. and i do not know what.
OK, you can rule out an issue with the polymerase. So, that means it's down to: DNA, primers, thermocycler conditions, the math you used for your Master Mix calculations, or your technique. Do your other PCR reactions work?
The primers would be made individually, it does not matter what the other ones are in the order.
A weak band means that everything CAN work, but it's not working particularly well. And 1000bp isn't a large product. I know it sounds counter-intuitive, but try diluting your DNA 1:10 and 1:100. If there are any PCR inhibitors in the DNA, then this can help get them at a low enough concentration to not interfere with amplification.
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