I am working on an undergraduate project within a group. The topic is to identify carbapenamase resistnace gene in K.pneumoniae and E.coli. The target genes are OXA-48, KPC, IMP and VIM. We are doing 2 seperate PCR: Simplex for OXA-48 gene, Multiplex for KPC, IMP and VIM gene. So far, the OXA-48, KPC and IMP primers are not giving products. Though the VIM gene primers are consistantly giving product. My peers are getting consistant product as well, all of thier primers are working, but not mine. I having a working solution of 20 micromolar, stored in 4oc fridge. We are using the sigma ReadyMix kit for the simplex and Qiagen Multiplex kit for the multiplex. Could someone plase put me out of my misery and try to explain why my primers are not working? Thank-you!!!
The PCR conditions had been optimized
For OXA-48 simplex: 94-10 min; (94-40s, 57-40s, 72-1min) 30 cycle; 72-7min
For Multiplex: 94-10 min; (94-40s, 55-40s, 72-1min) 30 cycle; 72-7min
We are using published primers: Article Development of a set of multiplex PCR assays for the detecti...
Since your colleagues are successful in getting product, you could ask them for a small aliquot of their primers to try them with your conditions. You can also ask them to try your primers with their conditions. That way you would know if the primers are the problem. If you don't get product even with their primers, you can check one by one the other elements. I agree with Daniel in that primers should not be stored at 4°C.
Hi,
It could be degradation of primers at 4°C (-> new working dilution), excess salt in your extracts inhibiting the reaction (-> dilute an aliquot for PCR), not enough mixing when assembling the PCR reaction (especially if you add a small volume of template DNA to a distributed mastermix)
Good luck
Daniel
Since your colleagues are successful in getting product, you could ask them for a small aliquot of their primers to try them with your conditions. You can also ask them to try your primers with their conditions. That way you would know if the primers are the problem. If you don't get product even with their primers, you can check one by one the other elements. I agree with Daniel in that primers should not be stored at 4°C.
First you need to observe your gel properly:
a) is it is blank with no bands - any of your PCR reagent could be cause of your problem,
b) however if you are getting a primer dimer which you might get below 100bp band then your primers are fine, problem is with the PCR reaction/reagent.
I'm assuming the problem is the first one then any one of the below could be the issue:
a) storing working primer stock in 4°C for long duration and freeze-thawing is not a good idea-it degrades your primers. Store them in -20 and thaw on ice always and keep it back asap after usage.
b) I agree with Francisco that you need to change one reagent at a time to find out what is not working. If all reagents I fine then I would suggest you to look at your primer seq once again, sometimes high GC content or repeat sequences make it impossible for the primer to bind to your template- you can either try touch down PCR to increase sensitivity and specificity or you may want to redesign your primer keeping GC content below 40% and checking self-annealing score etc
c)Sometimes high GC content in your template seq causes difficulty in denaturing
If thats the case, you can try hotstart PCR ( u can do manual if you don't have hotstart Taq pol by not adding enzyme at the beginning of PCR and pausing your PCR reaction after initial denaturation of 5 mins and then adding your enzyme so that your template gets enough time to denature without simultaneously starting amplification)
At last, do not worry, it happens to all of us.
Good luck
Anuja
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