How to cut PCR product with restriction enzyme ?
mdbcs001 at cmu.chiangmai.ac.th
Fri May 27 02:58:43 EST 1994
May anyone tell me how to cut the PCR product with restriction enzyme?
Recently I have performed the PCR of a gene and then I want to detect the
point mutation in the product with a restriction enzyme. I have tried to
cut it directly from the mixture but there are some problems. First, I
used the reaction mixture of 50 microlitre and it was too much to be loaded
in the slab gel for further detection by electrophoresis. So I applied
half of them instead and found that the band was almost unperceivable and
also it was not cut completely. Do I have to extract the PCR product out
of the reaction mixture before cutting with the restriction enzyme ? I
really do not want to do that because it takes time and may lose some
The second question is how to adjust the reaction volume for
restriction enzyme cutting . In the definition of restriction enzyme, one
unit of restriction enzyme is the efficacy of cutting the DNA of 1
microgram in 50 microliter in one hours. If I want to cut 1 microgram of
DNA in 20 microliter, how can I do ?
Thank you in advance for all the respond and suggestion.
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