troobleshoting for colonies background - (May/02/2006 )

hi all


i need a reason for my experiment i try to cloning qwiz with insert(800bp) when i digested with sal1 & Ecorv then ligation i got to much colony of backgrounfd and the insert not ligated with the vector i try several time with long incubation for digestion and ligation so can any one help me



thank you

could you please give more details?

exactly what time(s) have you done the digestions? under what conditions? (sequential, or double-digest?)

how are you purifying, and at what steps?

at what steps do you look at your fragment and vector via gel?

where do you get your fragment?

could you please tell us more detail?

exactly what time(s) have you done the digestions?

around 2 hrs

under what conditions? (sequential, or double-digest?)

double-digest

how are you purifying, and at what steps?

with Purification of DNA by phenol extraction and ethanol precipitation

at what steps do you look at your fragment and vector via gel?

i don't look at it on gel

where do you get your fragment?

from gwiz-GFP plasmid

by fragment, I meant insert, sorry!

If you don't ever look at your vector on a gel, it is very difficult to pinpoint the problem. it is likely there is an issue with digestion, or you are losing some of your sample somewhere