No Bands After PCR

[labratty]

Hi,
i am not getting any pcr bands on agarose gel electrophoresis!!

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

[kmunson779]

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]

If the suggestions by kmunson dont work Change the water you are using!!!! may be that would work

[polymerase1]

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]

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.

[Brotec]

Maybe forgotten to add one of the pivotal PCR ingredients??? Forgetting to add Taq Polymerase always give no yield