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PCR inconsistency

PCR

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#1 chachick

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Posted 19 October 2011 - 11:17 PM

Hi guys,


I am new here. Now I am facing a problem in PCR. My PI asked me to repeat the PCR that my colleague have failed.
I got everything new, except the cDNA template. The primers are newly designed.



I try to optimize the PCR by using the positive control. The problem is, I can get the right band (ard 900bps) the first 2 times, and then when I run my samples with positive control, my samples have single band but no band for positive control.



I have tried it like 3 times and every time using new aliquots. This is exactly what my colleague had encountered and it happens to me now. My PI told me that this is his baseline and I am very frustrated.


Please can anyone help?

#2 allynspear

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Posted 20 October 2011 - 04:25 AM

Are you doing anything different when you run your samples? This may be the case of trying to do too many samples at once. If possible, I would pick one or two samples that you suspect are positive, and set up your PCR with those two samples, as well as a positive and negative control.

If you don't think this is the issue, you can provide a little more detail about how you set up your sample PCRs and we can try to troubleshoot.

Best of Luck.

#3 chachick

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Posted 20 October 2011 - 04:53 PM

I am using Qiagen Hotstart Taq. I juz mix dNTP, buffer, taq, primers, and dH2O into a mastermix and aliquot to 12 tubes, which includes the positive and negative control. Then I add my templates.


I am trying to check if the cDNA is degrading today, wish me gd luck.





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