Best way to purify PCR product for sequencing - (Nov/04/2005 )
I just want to ask your routine and best way to purify the sample for dna sequencing? ( advantages and disadvantages based on your experiences)
1. should i splice the band from my 20 ul pcr product and purify it later
2. amplify the target gene individually again ( coz am using multiplex) using 20 ul or more reaction volume and purify the pcr product after checking it in agarose gel ( 5ul pcr product loaded in agarose gel)
Cutting a band from a gel is a good idea, especially if you are sequencing using one of your PCR primers. Otherwise, even nearly invisible amounts of primer-dimer can poison your sequencing reaction. Since the primer-dimers are short, they don't weigh much, can be nearly invisible, but have many molecules of sequence matching your primer.
Since your are runing multiplex PCR, you have to cut each and every single band out from gel and purify them seperately. Only then can send for sequencing.