Cutting a PCR-product

alex dobrovic adobrovi at medicine.adelaide.edu.au
Wed Aug 12 17:59:09 EST 1998


We do this all the time. Take 10ul of PCR product and 1ul of RE (5-20U/ul),
2ul 10x RE buffer and water  up to 20ul with and incubate.

In some cases, the PCR buffer may influence the cutting but this is not
generally serious.

Perhaps in some cases, you could look at adding another ul of PCR buffer
rather than 10x buffer or 1ul of 10x RE buffer but we generally add 2 ul of
10x RE buffer.

>> Hi,
>>
>> I want to confirm the sequence of my PCR-product with a digestion. Can I
>> perform the
>> reaction directly, only adding RE and the buffer for RE or do I have to
>> extract the DNA from the PCR-mix.
>>
>> Thanks in advance,
>>
>> Chris
>
>Chris:
>It may depend some on the restriction enzyme, but yes -- you can often
>directly digest the PCR product as you suggest, although I have found
>that you have to hit it pretty hard (up the RE units by at least a
>factor of 5).  I published a paper a several years ago describing the
>technique (Spinella, D.G., 1990, "A rapid method for genotyping mice for
>T-cell receptor VBa and VBb haplotypes by PCR analysis of whole blood"
>J. Immunol. Meth. 135: 209-212).  I'll send you a reprint if you like.
>Cheers. --Dom Spinella


Alexander Dobrovic, Ph.D.
Chief Medical Scientist
Department of Haematology-Oncology
The Queen Elizabeth Hospital
Woodville, SA 5011, Australia

Affiliate Senior Lecturer
Department of Medicine
University of Adelaide

Tel 61-8-8222 7676 (new as from April 1998)
Fax 61-8-8222 6046
adobrovi at medicine.adelaide.edu.au





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