Hello everyone,
there are various restriction enzyme for us to select for the best outcome of RFLP. In general , we will choose the restriction enzyme which only have one restriction cutting site; however, if there are more than one types of restriction enzymes that all have only one restriction cutting site, what else standard can we apply to choose the most optimal restriction enzyme?
FOR EXAMPLE:
FauI, BstUI, Acil,all of them have only one cutting site for the sequence GAACCCGCGTCATACC, what standard can we apply to choose the best one for RFLP?
Hi there,
Choose the less "exotic" one. I mean the one having the more standard properties.
I would usually choose the cheapest unless there was one that cut at high temperature not 37c for speed and ideally one that many companies sell as this may mean it is a reliable cutter. Check the New England Biolabs web site to see if the enzyme has star activity because all of your product may have the cut site it now occurs every 300bases not every 4exp6 bases perhaps in the genome so you may have to add more enzyme to cut completely which means adding more glycerol which may cause star activity. More specifically in your case the best enzyme does not produce another fragment of the same size as one of the informative fragments but ideally does have another cut site in your amplimer giving a visible fragment that proves that the enzyme is cutting for confidence in the situation where you get homozygous non cutting at the informative site
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