We extracted frog RNA to obtain its peptide sequences. However, after retrotranscription all the products were truncated. We used a universal polyA to perform the first round and one specific primer and a polyA primer to perform the second round. After cloning with TOPO and sequencing with M13 primers, we found out that the primers were delimiting very short sequences. It seems that the ployA primers didn´t bind at the polyA tail, but in the middle of the gene. How can we avoid that happening again?, thank you very much
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Problem with polyA retrotranscription: I have truncated products
Started by RanaRNA, May 11 2016 10:46 AM
cloning retrotranscription polyA TOPO PCR
1 reply to this topic
#2
Andrea Fortina
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Posted 20 June 2016 - 12:31 AM
How much time did you run your reaction? It is perhaps your reaction time not long enough?
Did dNTP enough for the retrotranscription?
Did you run DNA gel to test your production,how about your result?
Andrea Fortina
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CreativeBiomart, Recombinant protein expert
Also tagged with one or more of these keywords: cloning, retrotranscription, polyA, TOPO, PCR
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