Dear collegues!
we extracted RNA from apple peel. We need clean RNA without DNA contamination, so we check our RNA samples by PCR. Samples are fresh. and reagents are the same. BUT one time PCR works another time - no band in DNA control. Only thing we change is the electrophoresis chamber. We have no idea what can it be? Do you have such a problem? Please write your suggestions about this. Thank you in advance. looking forward to hearing from you soon.
I have had this kind of problem in my lab, but usually its has been related to distraction during the preparation. Avoid any kind of distraction and make duplicates of each PCR.
Are you maintaining the reactives in ice during the preparation of the PCR? are you using a mastermix or a separated component PCR kit? Mastermix PCR kits are usually better for rutinary PCRs, since they have less steps to prepare it and are faster.
I believe you are facing no results in PCR, maybe the template DNA is less/not present. Did you check the concentration before use? and If you are using kits kindly check if it is in working condition.
If you are not getting PCR results it may be due to various reasons. You could troubleshoot it one by one, but it will be a tedious process.
My guess is that the Template is not enough.
Good luck.!!
Did you also include a DNA marker lane and saw it ran down nicely and with a good result? Did you remember to add EtBr?
Dear Lidia, I suppose we all need some more details. What's your DNA positive control source? Why don't you DNAse your RNA? When you do not see the control's band, do you see samples' bands? How many cycles do you run? What reagents do you use? Some Russian kits require more TaqPol in the reaction than it's stated in their protocol. And I agree with Yuan-Yeu, are you sure your electrophoresis works?
Hello again, thank you for your comments,
here I answer to your questions:
David, yes sure we are maintaning all reactives on ice during mastermix preparation from separated conponents.
Mohamed, we have both positive control - DNA, and negative control (mastermix). Positive control is defenetelly OK.
Yuan-Yeu Yau, yes we also Include DNA marker in gel, and it works good, we never forgot to add EtBr.
Peter, we have treated our RNA samples with DNAse, and we do these PCRs to check is our RNA really without DNA contamination. We run tested PCR programm, which defenetelly OK. We use reagents for MasterMix: water, 10x PCR Buffer, MgCl2, dNTPs, Primer Fw, Primer Rv, Taq. This is not Russian kit, we work in Germany.
So dear collegues here is example for you to imagine: yesterday morning we take 18 RNA samp+positive control (DNA)_ negative control (mastermix) and did PCR. Results are fine - we had a band in positive control. After that we took THE SAME reagents and did PCR with 40 samples and controlls - Result - NO band in positive control. Then another person tried - also no band. But the day before we checked our positive control with the same reagents - PCR worked. This is unexplicable...do you have some ideas?
Have you already tried changing all the PCR reagents? Is there any connection with the duration of the PCR setup? For example as you say 18 samples setup did its best, while the 40 samples one did not work out.
Did you and your co-worker use the same PCR machine? If yes, does the PCR machine reliable?
As Peter said, it is possible that the problem is in the number of samples, if you are making a lot tubes at the same time there are some problems that could be affecting your PCR. When do you add the polymerase to the tube? Polymerases usually have exonuclease activity, so it should be possible that during the preparation of so many tube polymerase was destroying the primers. Is the polymerase a hotstart enzyme?
If it isn't a complicate PCR, it should be useful to change the PCR kit and try with a mastermix all in one tube. This way, you only have to add water, primers, template and mastermix. More steps, more time, more possibilities to make mistakes, easier to fail.
Dear collegues,
thank you for your comments.
Peter, we prepare mastermix in 1 tube, add polimerase in the ed, then spread mastermix in pcr tubes, then add samples. Everything is located on ice during the process.
David, Polymerase is hotstart.
Yuan-Yeu, PCR machine is reliable and programmir right tested many times.
so mayby you are right...Next week maybe we will try to change PCR components and run only 18 samples again.
thank you again.
regards,
Lidia
Hello, Lidia.
I would suggest scaling up the number of samples. It could be because of human error, since you mention that everything else is working. Start with your controls plus 3 samples. If it works fine, then try again with a higher number of samples. At the point Where problems start showing up again, some adjustments in the way you are preparing the reactions might be needed, so that you can continue scaling up.
Dear friends,
thank you for your kind participation in our problem. We have tried 6 samples and DNA in control appeared!
later will try with more samples.
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