PCR - RFLP of plant cpDNA - Can I use several restriction enzyms together? (Sep/27/2005 )
Hi! would you answer for newborned. I am going to look for genetic markers using PCR with universal primers (first step of the work) for noncoding region of plant cpDNA. Next step is digesting of the PCR products with different restriction enzymes. I would like to add several restriction enzymes together to pick up screening of polymorphism of every PCR-product ( fragments DNA ). Is it realizable? I don't see such way in the press. 
-panas-
Hi
Typically if you are looking at RFLPs you would only use one or two restriction enzymes per reaction, so as to get a unique pattern for each. If you use lots of restriction enzymes you will end up with a huge number of small fragments that will be hard to analyse.
Bob
-bob1-
QUOTE (bob1 @ Sep 28 2005, 03:46 AM)
Hi
Typically if you are looking at RFLPs you would only use one or two restriction enzymes per reaction, so as to get a unique pattern for each. If you use lots of restriction enzymes you will end up with a huge number of small fragments that will be hard to analyse.
Bob
Typically if you are looking at RFLPs you would only use one or two restriction enzymes per reaction, so as to get a unique pattern for each. If you use lots of restriction enzymes you will end up with a huge number of small fragments that will be hard to analyse.
Bob
Thank you. In case of chloroplast DNA, PCR products are fragments of 400-1700b.p. having one -two restriction site or don't having one. So I need to check a lot of sample+primer=product/enzyme combinations to find variations caused by insertion/delition. It is not huge number of combinations, may be 10 for each product according to restriction enzymes number I check. I thought the only save time and chemicals. If is any reason not to do this one stock reaction?
-panas-