low pcr product
Posted 10 February 2012 - 04:16 AM
Posted 10 February 2012 - 05:55 AM
In my experience is already happened that the secondary PCR performed on the product of the first one with the same primers does not work well. Sometimes I got smear and sometimes no bands completely.
In my opinion sometimes can occur, during PCR, a short deletion at the ends of the product and this does not allow the annealing of the same primers again.
Why do not you try to increase the cycles number (5 are enough) during the first PCR to get more DNA product??
Posted 10 February 2012 - 01:36 PM
I never trust anything that can't be doubted.
Posted 17 February 2012 - 05:39 AM
but with double band, and re amplify again , got easier this time and time and purify( if you have multiple band)...
could be your gene has high GC content? or your primer responsible for that...secondary annealing maybe?
but DMSO can help to solve this yeah!