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Problem related to Bcl-2 amplification and sequencing

Bcl-2 sequencing

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#1 litlbee

litlbee

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Posted 01 May 2012 - 02:54 AM

Hi everyone.
I hv been trying to amplify dog bcl-2 gene since a long time and have tried its pcr amplification with a long list of primers but got luck only with one primer set which amplify a region within the ORF. There are only two Bcl-2 ORF sequences reported in dog. Sequencing results of this amplified product are very frustrating!! They match 99% with chicken Bcl-2 and only 42% with dog. At first place i thought it might be due to some contamination with chicken DNA. Then next time i took all precautions and used everything new from plasticware to all chemicals for RNA/DNA extraction & PCR. This time i amplified gene from both cDNA and DNA but after cloning and sequencing I am getting the same results i.e. 99% similarity with chicken gene.
Has anybody faced the same problem with Bcl-2 amplification or sequencing results???

Plzz suggest something!!!





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