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Help need in 5' RACE PCR

RACE PCR Invitrogen clontech

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#1 mebrahtu

mebrahtu

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Posted 24 August 2011 - 10:37 AM

Hi all,
I am performing RACE PCR using the clontech kit (smarter RACE cDNA amplification). The 3’ RACE PCR work very well and amplicons were produced both in the internal control (using GSP1 and GSP2 primers) and 3’ RACE sample (using GSP2 and UPM primers). However, in the 5’ RACE PCR only internal control (using GSP1 and GSP2 primers) produced amplicon but not 5’ RACE sample (using GSP1 and UPM primers). The fact that 3’ and 5’ internal controls produced amplicons (the same size) tells me that GSP1 is working fine but I couldn’t find any explanation why 5’ RACE sample is not producing an amplicon.
I also tried the invitrogen kit (RACE system for rapid amplification of cDNA ends, version 2.0). The 3’ RACE PCR produced amplicon but not the 5’ RACE PCR and I am puzzled by that.
I am running of time and I would like to hear your suggestions and inputs. Thanks

meb





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