mRNA vs ds cDNA - Size differenses between mRNA and ds cDNA (Feb/21/2008 )
please help me understand following results:
i amplified 2ug of fungal mRNA (size range on gel 3-0.5kb, as suppose to be) with SuperScript™ II Reverse Transcriptase (1st strand) and with mix of DNA polymerase I, RNase H, E.coli DNA Ligase for 2nd strand. However ds cDNA appears to be between 6kb and 0.4kb.
I've never saw such a difference.
Appreciate any help
Not sure what the ligase is for. Are you trying to make plasmids out of your cDNAs? Perhaps the ligase is ligating a couple of your cDNAs together end to end.
E. coli DNA polymerase shouldn't be able to ligate blunt ends, however this could happen under certain conditions (e.g. presence of PEG in your reaction etc). Could it be that the conditions you used caused E. coli DNA ligase to join blunt-ended fragments, producing bigger sizes than expected?
Thanks for the reply.
The 2nd strand enzymes mix is a part of SSH kit, so it's a standard procedure (no PEG is present in the reaction) and it's never happened before. Do you have any other ideas why this could happen?
Thank you so much.