How to cut PCR product with restriction enzyme ?

Wirote Tuntiwechapikul 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 
product.
	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.
					Wirote.




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