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RT-PCR primer dimers and cDNA degradation


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#1 koralreef3516

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Posted 18 June 2009 - 06:41 AM

Hello,

I have been trying for a while now to perform two-step RT-PCR. We are looking at e-cadherin, alpha smooth muscle actin and b-actin. Both b-actin and e-cadherin have worked and we have amplified both. But everytime we try to amplify alpha smooth muscle actin we get a primer dimer. I don't know how to remedy this. Also when we run the cDNAon a gel it is degraded. How can I prevent the cDNA from degrading? Is this the reason for the primer dimer? We treat our samples with DNase so iwas thinking that maybe degrading the cDNA but even when we use untreated RNA in our reaction the cDNA is degraded. Any suggestions would be greatly appreciated.

#2 pcrman

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Posted 18 June 2009 - 02:23 PM

How do you know your cDNA is degraded? On agarose gel, cDNA appear as a smear ranging from several KB to hundred bp. If two other primers worked, then the dimer is mostly caused by self-pairing of the primers.

#3 littleaxt

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Posted 23 June 2009 - 04:04 AM

Hello,

I have been trying for a while now to perform two-step RT-PCR. We are looking at e-cadherin, alpha smooth muscle actin and b-actin. Both b-actin and e-cadherin have worked and we have amplified both. But everytime we try to amplify alpha smooth muscle actin we get a primer dimer. I don't know how to remedy this. Also when we run the cDNAon a gel it is degraded. How can I prevent the cDNA from degrading? Is this the reason for the primer dimer? We treat our samples with DNase so iwas thinking that maybe degrading the cDNA but even when we use untreated RNA in our reaction the cDNA is degraded. Any suggestions would be greatly appreciated.


Hi!
Probably just bad primers that build dimers, so you have to design new ones. You can check them for producing dimers here: Beacon designer free




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