Hello to everyone- this is my first posting here. But I always got lot of very useful and valuable information from this forum.
I use the non-kit version of RNA seq protocol to generate RNA seq libraries. I was successful in the past. I used dynal beads, Ambion Frag buffer, got good mRNA, good fragments (bioanalyzer), good double stranded cDNA (bioanalyzer, high sensitivity DNA chip). I also see good smear after my ligation on the gel. But I've been getting extremely bad PCR results. Very low, strange bands. Not sure where in the next 3 steps after double stranded cDNA synthesis- 1). End repair, 2). A-base addition, or in 3).Ligation itself (hard to believe) the problem is happening, but I'm losing it all. It is not PCR, because my positive control worked. I've using a protocol similar to the one in http://www.unc.edu/htsf/files/PM%20p...%20DNA_1.0.pdf
If anyone used the custom protocol as this one, What is your instinct about where I'm making it wrong? If you have encountered similar problem in the past, how did you fix it? I have been using the buffers that come with the NEB enzymes for that given reaction- for the end repair, 50%-50% of T4PNK buffer and Buffer 2.
thank you. SP
I use the non-kit version of RNA seq protocol to generate RNA seq libraries. I was successful in the past. I used dynal beads, Ambion Frag buffer, got good mRNA, good fragments (bioanalyzer), good double stranded cDNA (bioanalyzer, high sensitivity DNA chip). I also see good smear after my ligation on the gel. But I've been getting extremely bad PCR results. Very low, strange bands. Not sure where in the next 3 steps after double stranded cDNA synthesis- 1). End repair, 2). A-base addition, or in 3).Ligation itself (hard to believe) the problem is happening, but I'm losing it all. It is not PCR, because my positive control worked. I've using a protocol similar to the one in http://www.unc.edu/htsf/files/PM%20p...%20DNA_1.0.pdf
If anyone used the custom protocol as this one, What is your instinct about where I'm making it wrong? If you have encountered similar problem in the past, how did you fix it? I have been using the buffers that come with the NEB enzymes for that given reaction- for the end repair, 50%-50% of T4PNK buffer and Buffer 2.
thank you. SP
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