Hello everyone,
I'm trying to do a PCR on unknown cDNA fragments from a RNA immunoprecipation experiment in order to sequence those fragments. My question/problem is how would I do this since I don't have sequence specific primers, my only thought was to do a cDNA or genomic library. I'm also using random hexamer primers for my RT-PCR to amplify all RNA associated to my protein. I was wondering if there was a easier way to proceed.
Any helpful ideas are appreciated
Thanks
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How to amplify unknown cDNA sequences from a RIP for sequencing
Started by Sebastien, Jan 07 2010 05:35 AM
1 reply to this topic
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Posted 18 February 2010 - 01:34 AM
Hi,
If I were you, I would design a primer with a d(T)16-20 (say, primer P1-Tn) and use this primer to synthesis cDNA via RT. After that, I would purify the cDNA (use something like Qiagen Mini Elute kit) and ligate all cDNA frgaments with a 5'-phosphorylated, locked 3'-end oligo (say, adapter pA1, randomly unique sequence) using T4 RNA ligase kit (reaction should be long, 18-24 h at 22ºC). Then I design another primer (say, primer A1-P) that complementary to the adapter pA1. Amplification of the whole cDNA ibrary with these two primers, P1-Tn and A1-P. The PCR product is purified and cloned. All positive clones are sequenced and you may get the genes you look for.
I hope this may help you.
If I were you, I would design a primer with a d(T)16-20 (say, primer P1-Tn) and use this primer to synthesis cDNA via RT. After that, I would purify the cDNA (use something like Qiagen Mini Elute kit) and ligate all cDNA frgaments with a 5'-phosphorylated, locked 3'-end oligo (say, adapter pA1, randomly unique sequence) using T4 RNA ligase kit (reaction should be long, 18-24 h at 22ºC). Then I design another primer (say, primer A1-P) that complementary to the adapter pA1. Amplification of the whole cDNA ibrary with these two primers, P1-Tn and A1-P. The PCR product is purified and cloned. All positive clones are sequenced and you may get the genes you look for.
I hope this may help you.
