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3' RACE PCR from RNA without poly A tail

3' RACE PCR from RNA without poly A tail - Molecular Biology Techniques

3' RACE PCR from RNA without poly A tail - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 08-09-2012, 04:14 PM
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Default 3' RACE PCR from RNA without poly A tail



Hi guys, I am new in this forum and I hope you guys can give me some suggestions regarding 3' RACE PCR. I been doing a lot of RACE PCR nowadays using Clontech. I have no problem generating the 5' RACE PCR and also getting good sequences after cloning. My problem lies in 3' RACE PCR. Since my RNA doesnt contain poly A tail, I have to use a kit to add this. Afterwards I use it directly to do 3'RACE PCR. After running usually I don't get a band or the band is too weak. I tried increasing the amount of RNA that I put in making the cDNA for 3' RACE PCR, and in a way it also increase the concentration of the cDNA and I got better bands than before. But during sequencing although its inserted, it is not properly read during sequencing. They said that after the poly A tail, the sequence appears to be smeared or something to do with enzyme slippage. can anybody suggest how to prevent this? I was able to get some sequences for my 3' RACE PCR but still a lot of the samples did not have good results. Any suggestions will be much appreciated. Thank you. (Sorry guys my story was to long^^)
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pcr , poly , race , rna , tail


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