Digesting PCR products - (Apr/27/2006 )
HELP! I'm currently a student in a molecular diagnostics class, working on her final exam. We were given a gene with a noted polymorphism in which to design an assay around. So my plan is to amplify by pcr then digest with a restriction enzyme, then run it on agar. My problem is this: we need to show the quantities of reagents we will need. I know that the amount of RE needed is based on the amount of DNA in the sample, but I"m not sure of how to figure out how much DNA I will have after amplification. ANy help would definately be appreciated! Also, do I have to run my digestion separately? Or can I include that enzyme in with my PCR? I was under the impression that I had to digest after the PCR was done....
Please, please, help!!!! 
so... well many questions so i will tell as i do generally.
I amplify by pcr a fragment generally in 50µl.
I analyse 1 to 4 µl depends on the pcr enzyme. For taq it's 4.
For digesting enzyme, you'll have to face 2 major points :
if your enzymes shows star activity, you have to purify your pcr product in order to resuspend DNA in the buffer recommended for the enzyme.
If you purify the product, i resuspend buffer generally in 85µl
I add 10µl of a 10fold concentrated buffer, ad 4µl of enzyme and 1µl of a 100X BSA (does not interfers with digestion but protects the enzyme)
Thank you for your help! I was very stuck. The links were great.