Restriction digestion of PCR product - (Nov/16/2006 )
I'm a new student. I'm not getting the results for my restriction enzyme analysis.my enzyme, Taq I is from NEBiolabs,incubated for 4 hours at 65 degree.I'm using PCR product for restriction analysis.
The components I used for my reaction are
DNA - 5
BSA & buffer is provided along with the enzyme.I'm new to this field & I don't know what is the problem with it.Please help me.
Your reaction conditions are all wrong. Try these:
DNA: 5 ul (or whatever will give you 500 ng - 1 ug or so)
Buffer: 5 ul of the NEB Buffer 3 (stock is 10x, so for a 50 ul reaction, you need 5 ul)
BSA: 0.5 ul (stock is 100x, so for a 50 ul reaction you need 0.5 ul)
Water: 38.5 ul
Enzyme: TaqI 1 ul (1 ul should be plenty, and total volume must be less than 10% or 5 ul for a 50 ul reaction)
Total volume 50 ul.
Incubate at 65C for 1 hour, heat kill at 80C for 20 minutes.
(this is unusual, most enzymes work well at 37C)
In general it is a bad idea to try to use low volumes for cutting.
Your DNA should be a small component (10% in this case) of the volume of your reaction, which helps dilute and avoid problems in the composition of the DNA buffer or contaminants.
did you purify your PCR products before digestion?
I believe sreeja intends to use the PCR product for diagnostic purposes only. If that is the case, as long as the PCR mix is diluted enough by additional water and RE buffer, it shouldn't matter to much if the PCR product has not been cleaned.