I have real problem working with PCR. For example, one day my positive control has a band after gel doc but in next day this same control hasn't any bond and the other bands are very faint. I am so confused about this. Should I change my materials (Taq, buffer, Mgcl2)?
What do you think?? Please help me. Thanks.
Hi, did you change the electrophoresis buffer? To less EtBr in your gel? Cheers, Nadine
I think, now You can change all Your working solutions!
if the same problem repeat - probably you have contamination in the template!
I think you should change the working solutions... if the problem was in template it should given band in next day same control...
all the best
Having bands means that your PCR worked under the given conditions. You have not mentioned in your question, whether you changed any of the settings (ie temperature, number of cycles) in your latter PCR runs. If you did, then positive control (PC) may not work because the reason you chose a certain template as a PC, is because you know it would amplify. If you, however had drastic changes in your PCR program, then it may affect the amplification success. Ideally, you could try rerun the PCR with new ingredients. Good luck!
Did you Change the volume of the reaction ? This can Change your results as well, as the Dynamics is different in a different volume (Molecular crowding etc etc)
Even I have encountered similar problem in my previous lab.See PCR is very sensitive technique.A small variation in your pcr conditions will end up giving you no bands which worked absolutely fine previously.Make sure that your template is still working ie,not degraded.The sterile water which you use to dilute your cDNA,primers,MgCl2,buffer and your pcr reaction itself must be nuclease free.Always use a fresh vial of sterile H2O for your pcr reactions.Also be sure of pipetting efficiency ,you will be adding very low volumes of your Taq polymerase ,so try to avoid pipetting errors.Look through the tube and dispense your components.And always follow the order of adding H2O,Buffer,MgCl2,primers,Taq and finally your template.Pipette up your entire reaction mixture 5 to 6 times or a brief vortex which will ensure proper mixing of your components.Follow this and I am sure you can troubleshoot the problem.Also keep in mind to store your components at the recommmended temperatures for their proper stability and activity.
Please remember, the concentration of MgCl2 is critical in PCR. That's why the PCR kit provide separate Buffer and MgCl2. Try different [MgCl2] in your reaction. It could be a "miracle". Good luck.
Its better to use fresh pcr components. Check the protocol you are following at least annealing temperature. Good Luck...
Reproducibility of the results needs a bit of practice in the beginning.
A common error, strange but true, that I have noticed done by the beginners is pipetting error. This leads to problems in reproducibility. Also have the exact protocol written down and have total concentration on the work to be done. Tick the PCR mix reagents on your work book one by one when you add them.
Make sure you are adding right proportion of DNA into the PCR tube.
You can ask your senior to perform PCR once in front of you and observe carefully.
Make sure you know how to pipette. Rest of the things are MgCl2 concentration and annealing temperature you need to work on.
It could be also the electrophoresis problems, such as gel concentration or EtBr.
Avoid repeated freezing and thawing of PCR reagents, aliquot in separate tubes.
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