i have standardized a PCR condition, it worked well for two to three run, later again it is not working, need to standardize again and it repeated thrice. what might be the possible reason for this?
we have tried all the possible trouble shooting ( change of concentration of DNA, increasing primer volume, new primer dilution, change of master mix vial. gradient temperature changed)
seems like an inhibitor got into your reaction somewhere along the way. make new stocks of primers and use a new stock of polymerase to see it that helps.
Here are few suggestions.
1. I would suggest make new dilutions of your template DNA and primers.
2. Make sure you are operating the PCR at optimized temperature. I hope you have done gradient PCR already if so try a little stringent condition.
3. Use new Master mix. Be careful about master mix ,excessive thawing and freezing multiple times can make it less effective and may make taq inactive.
4. Must run positive control and negative control with your samples to check for any contamination.
5. Also check you are adding correct concentration of template DNA and primer you used for standardizing. If you are making new dilution with new ( different)concentrations. You need to optimize from scratch through gradient PCR.
6. Pipette very carefully sometimes all the regents dont mix well and may lead to negative results. Try doing a short spin after making aliquots in PCR tube.
Hope this helps
Good luck.
Hello,
This issue might be due to one or a collective of reasons such as storage conditions, contamination, spontaneous oligomerization of the primers...etc.
Thus, we need more information to be able to properly help you.
information like storage conditions, primer dilutions, did you dilute the primers into ready-to-use aliquots instead of using the main primer tube? template DNA concentration, MMX storage and expiry date, among others
All the best
Try less DNA instead of more. It could be the stuff in the lysis mastermix causing the problem instead of lack of dna.
Sana Shamshad is correct.
I'll emphasize the third point, as it may be your PCR's main issue.
Please divide your master mix, primer, and dH2O into 1.5 ml tubes.
1 ml from a master mix kit can be divided into 5 tubes (200 ul each).
One tube can be used for PCR while the other is kept frozen (You can use the next tube after finishing the first tube).
The same goes for primer and dH20.
This prevents contamination and over-repeated thawing and freezing of your working solution, which will affect the result.
I hope this will help you out.
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