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-   -   No Bands After PCR (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/3052-no-bands-after-pcr.html)

labratty 02-19-2008 06:11 PM

No Bands After PCR
 
Hi,
i am not getting any pcr bands on agarose gel electrophoresis!! :cussing:

what should i do??? i have tried all temperatures and optimization.... :cry:

kmunson779 02-19-2008 07:41 PM

Re: No Bands After PCR
 
Well, I'm not sure what "optimization" you've tried so here's some things I'd try:

1) PCR additives: DMSO, betaine
2) Less template: see if there's a PCR inhibitor in the template DNA (knowing what kind of DNA you're starting with would help troubleshooting)
3) Positive control: Do you have a known template your primers will amplify? Or other primers that you know amplify your template? Or some PCR product you can amplify to make sure the thermocycler is working properly each run?
4) New primers: sometimes primer sequences just don't work -- did you check for hairpin formation, homo- and hetero- dimerization, matched Tms, etc.? Or perhaps the primer synthesis didn't work properly. Order some new primers and try all combinations.
5) Make sure the gel is running correctly -- does your size ladder migrate and stain properly?

Priyanka 02-20-2008 09:40 AM

Re: No Bands After PCR
 
If the suggestions by kmunson dont work Change the water you are using!!!! may be that would work

polymerase1 04-18-2008 05:18 PM

Re: No Bands After PCR
 
plz post-up what did you do and what optimization have you tried so we'll be more helpful as the problem of not getting any bands might belong to punch or reasons. so plz put up whatever protocols you've used before so we can exclude unneccessary criteria.

thx

radioheadhead 04-23-2008 05:01 PM

Re: No Bands After PCR
 
are you using an oil overlay?

If not, make sure the heated lid is working and that it fits down snug on top of your PCR rxn tubes. This was giving me trouble on my new thermocycler, I wasn't putting the tubes in correctly and the top wasn't snug.:rant2:

Brotec 05-30-2008 11:57 AM

Re: No Bands After PCR
 
Maybe forgotten to add one of the pivotal PCR ingredients??? Forgetting to add Taq Polymerase always give no yield ;)

NATHANALANPATRICKROGERS 01-24-2009 10:55 AM

Re: No Bands After PCR
 
Quote:

Originally Posted by labratty (Post 7007)
Hi,
i am not getting any pcr bands on agarose gel electrophoresis!! :cussing:

what should i do??? i have tried all temperatures and optimization.... :cry:

Your initial DNA could in fact be nothing more than glorified moon dust :notworthy:

cat1082 01-28-2009 02:56 PM

Re: No Bands After PCR
 
I wasn't getting any bands with my PCRs until I re-amped it. I took 1ul of the PCR product and used it as template in a new PCR mix and that gave me product. I was adding primers to a total DNA extraction and my gene of interest was expressed at a very low level so any amplification that I did get was still too small to visualise on a gel.

Rafterman 01-29-2009 08:13 AM

Re: No Bands After PCR
 
Hey,
from my experience most of the time it is a problem related to the primers.You should check for dimerization errors or hairpin formation. If all this doesn't work, just order some new primers with different combinations as primers aren't that expensive you shouldn't hesitate too long.

cheers
Heidi


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