I am trying to amplify full-length of lncRNA (>2000 bp) that contains poly A tail for cloning into expression vector. However, due to the poly A tail in lncRNA sequence, the reverse primer that I designed contains poly T in the sequence and it causes more than 5-10 degree difference in Tm between forward and reverse primers even when i've included restriction sites in both primers. Any suggestions about this? I also wonder does the cloning of full-length lncRNA into expression vector need to contain poly-A or not?
Could you please give me a suggestion to do about this?
Thank you for all suggestions.
Hi,
For resolving this problem you can modify the oligo-dT primer used for the reverse transcription step. The oligo-dT primer should be slightly longer, with additional sequence towards the 5-prime, Which should also contain restriction sites for subsequent cloning. After the reverse transcription step, use a forward primer and a new reverse primer (complementary to the 5-prime end of Oligo-dT) to amplify and later clone the 2 kb fragment. This approach has been commonly used during cDNA library synthesis. It will be useful
I suggest follow Ajay's advice above and match your primer sequences for Tm by adjusting their length. Do the first 5 PCR cycles at a generous (lower) Tm and then raise the annealing temp to that of the full length primers. During the first few rounds of PCR cycles only the matching bases will be important. If you can match the Tm of just those parts too, you should find everything works well.
Ok are you sure you mean polyA-tail? or polyA-signal? Could you give us the the first and last 100bp of your lncRNA insert? Im just curious.
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