In order to do a cloning I am trying to do double digestion of my vector with Sph1 and Not1 enzymes , the sites are 14
bases apart. My question is - is it appropriate to do double digestion at a time with fast digest enzymes (Thermo) which mainly have a common universal buffer or should I go for sequential digestion with the enzymes? my concern is - as both the sites are nearby will be there any interference during the double digestion using both enzyme at a time?
Thank you
14 bases is more than enough you can safely do the double digest in FD buffer if both your enzymes are fast digest.
Hi,
I suggest it depends on the downstream experiment you have with your vector.
If you are just checking the clones confirmation then fast digestion with both the enzyme is fine and if you are using this vector for ligation I suggest you to digest sequential digestion is appropriate for cloning purpose.
Dear Ravi
i think that as Hanna told you, if both are available in version with same buffer you can certanilly perfora double digestion.
However i suggest to you to evaluate enzyme Free cloning approach as PIPE Cloning or in-fusion cloning which do not require to perform digestion steps.
if you are interested on my blog
https://proteocool.blogspot.com/?m=1
you can find more details about PIPE cloning
ciao
Manuele
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