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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!!!! unsure.gif


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 :

for first question, the answer is in the question. So if you don't know purify your pcr product.

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.