So I did a PCR today. I've tried to amplify a quite CG rich region from a plasmid pTriex1.1. My primers should anneal and form a 666 band, instead I get a band that migrates at about 300 - 400 kb mark.
Is this non specific amplification or y plasmid could have mutated?
Flabbergasted by the situation, to be honest.
Fwd primer Tm = 62C
Rvs primer Tm = 66 C.
Annealed at 65 using Q5 2xMM.
This never happened to me before. I got non specific amplification before. Still..i got the correct band with the non specific amplicon (diff. template and primer).
That's a nice bright band on your gel.
I'm assuming that those primers are specific and can't anneal anywhere else in that plasmid (double-check to be sure).
It's very possible that you don't have the correct plasmid, especially if you didn't get it directly from a stock center. It's really common for folks to assume/forget/mix up what plasmid is which.
Sequence the band & see what you amplified.
Katie A Burnette thank you for the answer! I have ”triple” checked! I’ve also cheked to see if my primers bind anywhere else in the plasmids I was using (maybe a mix). I did transform more than one. Although it is a cg rich sequence primers don’t seem to bind anywhere else. I did do a restriction digest and it seemed ok. But who knows? Maybe I‘d better get my hands on a new plasmid. Will try a 2 step pcr tomorrow to see if I get the same band...
if you are following the right procedure and doing it correctly, there are two possible reasons for having a small band?
1. amount of the sample load during gel electrophoresis might be too small
2. or might be primer dimers happens during PCR work. ( you can check if the faint band happens or not!)
and I think your band is ok, you do not need to worry about it!
Mohammed Fatih Rasul The amount of PCR reaction loaded has no influence on the band size, only the intensity. The product is too large & too bright to be a primer dimer.
Adrian Ionut Cepleanu-Pascu My bet is that the plasmid is missing some of the expected DNA & isn't the plasmid that you want/need.
A restriction digest is only accurate within about 10% of the band sizes. If you expect any fragments that are, say 2000bp, you can't really see the difference between 2000bp and 2200bp. Missing 200bp is not necessarily going to be obvious.
Katie A Burnette that is pretty much what i thought also. Thank you.
Have you tried setting up a gradient PCR with varying annealing temperatures ? Try higher temperatures as close as 5C below extension temperature (You can increase extension to 76C). Judging by the strong amplicon, if you are confident about your template then the best explanation for a wrong sized band is non specific priming.
And since your sequence is GC rich, try using Betaine or Dimethyl Sulfoxide in your reactions to prevent stem loop like secondary structures from forming. This could cause premature truncation of the amplicon and give a smaller band than expected. Also, you are probably aware of this, some PCR polymerase kits come with few vials of the PCR reaction buffers that are optimized for GC rich sequence amplification. In Q5 pol's case there is a GC enhancer. You can try setting up the reaction in that buffer/enhancer if you haven't before.
Hope this helps.
Praveesh Valissery thank you for your answer. I am currently using Q5 master mix which basically should do the trick for hard to amplify samples, including CG rich. Because of funds ... limitations, I chose one that should work with everything and it has proof reading. It's not as good as regular Q5 with enhancer but ... Instead of trying to troubleshoot the issue I would be better off picking a different promoter. I'm not that confident about the template, to be honest.
Adrian Ionut Cepleanu-Pascu Alright. Best of luck with your work.
There are several reasons why you are getting a smaller band.
1.If you have already double-checked all your reagents, repeat the reaction using fresh primers, fresh PCR water, etc. Clean your pipettes and make sure that there is no contamination of your master mix.
2. If your PCR product has a high GC content, over 65%, it is a good idea to optimize the annealing temperature by using a thermal gradient. I can see that your annealing temperature is already 65C and even though I agree with using a high temperature when GC is high, you can also try 5 degrees below the primer's melting temperature and see how it goes.
3. Another reason is that the template is degraded or contains inhibitors. For this, you can dilute the template and increase the cycles. It would be great if you have a control sample/reaction using a pure plasmid, your template, a negative sample, and water. That way you can definitely be sure that your primers are binding on the right site.
4.Check your primer concentration. If the concentration is too high, there is a good chance that the primers are binding nonspecifically to undesired sites or binding to each other.
5. I agree here with Katie A Burnette ...Whenever I find that my bands are different from what I am expecting, there is a high chance that the design of the primers is incorrect. If you are sure that they are binging where they are supposed to, you can re-order the primers (could be a manufacturer error) , move your primer a couple of basepairs, check the GC content, Tm and start all over again !!
Good luck!
Check your primer targets again, looks like a non-specific amplification. Perhaps can try touch down PCR which may help. Otherwise, try designing a new set of primers.
Check your plasmid there might be something wrong with it, maybe you used the wrong plasmid.
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