Plasmid DNA cleaning - (Feb/01/2005 )
i changed amino acids for two of my mutants using site directed mutagenesis, everything worked fine till sequencing started, even the commercial primers could not sequence beyond 200-300 bp, with some samples not giving any results, so could someone suggest simple n easy method to clean DNA after purifiaction using promega miniprep kit, cause the sequencing lab people cleaned and it worked.
I think the most efficient way of cleaning your DNA would be a phenol:chloroform:isoamyl alcohol purification and then a sodium acetate/ethanol precipitation.
Normally, miniprep kits come with an elution buffer that contains Tris and EDTA. These buffers can interfere with the sequencing reactions, and so I always elute in purified water prior to sequencing. I would recommed that you try this first, and if it doesnt work, then do the phenol chlorofom purification and precipitation.
Are you eluting in water or elution buffer?
i'm using promega miniprep kit and have observed two paricularities :
First it is very important to let your miniprep column air dry correctly and room temperature. At least thirty minutes air-drying are neccessary.
The second one is for the sequencing. I've observed that when i start qith miniprep concentration under 600ng/µl sequencing was bad . I think of contamination in my miniprep solution (ethanol, salts, and other possibilities).
I use the amersham sequencing kit for my preps and before resuspending my sequences (in high deionized formamide), the drying must be on room temperature !!!
hope that could help you.