Primer extension problem - there are too many bands!! (Aug/26/2005 )
I have a difficulty to find out the transcriptional start point of a E.coli gene by primer extension.
There is no disticnt band, but a set of bands all over the sequencing gel, as in DNase footprinting.
Whan can i do to improve this?
It could mean multiple transcriptional start sites (TSS) or non-specific binding. Make sure your RNA is not degraded by checking it using gel analysis. Your primer should be at least 20 mer in length, binds at a position at least 100 bp downstream the TSS, has no significant homology with other mRNA sequences.