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PCR - No bands anymore in well functioning PCR


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6 replies to this topic

#1 Lea Lewin

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Posted 21 December 2004 - 06:10 AM

In our lab we do a lot of genotyping PCR of transgenic mice. Since 2 months two of our PCRs donīt work anymore. No bands at all (sometimes even the positive control is very weak or disappeares) , I really checked everything I could think of: new primers, same primers from different company, new dNTP, new dilution of dNTP, new Taq, HPLC-water new batch, DNA-isolation with different methods, OD for DNA concentration. The un-genotyped mice are piling up and my problem grows. Does have anybody any more suggestions! :huh:

Thanks in advance
Lea

#2 littlecell

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Posted 21 December 2004 - 08:19 AM

use another PCR machine!

#3 vetticus3

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Posted 21 December 2004 - 01:24 PM

blood sacrifice.

or play with the Mg concentration... or alter the temps just a little (gradient machine)... try using a fresh batch of buffers and enzyme... let someone else make it up... put in more DNA.... put in less DNA...

Edited by vetticus3, 21 December 2004 - 01:30 PM.


#4 Lea Lewin

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Posted 22 December 2004 - 11:04 AM

We already used 2 other PCR machines and a gradient cycler and all new batches of buffers, dNTPs and a new batch from the old Taq as well as a new Taq supplier and even an PCR enhancer.
Maybe playing with DNA amount can help.

Thanks a lot for the recommendations. :huh:

Lea

#5 scientist

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Posted 27 December 2004 - 11:21 AM

Did you change the method of DNA extraction?
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#6 ravibiot

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Posted 02 January 2005 - 10:53 PM

hey

do you check whether heating of buffer in electrophoresis, if so change the buffers regularly and try one more time.
Is your EtBr is working properly, are exposed much time to light

regards

#7 peixumol

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Posted 02 January 2005 - 11:16 PM

hi,we had met the same problem in our lsb too!we sloved by using PCR additives(BSA,DMSO,Tritonx-100,etc),why not have a try? :o
paul in NanJing China
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