I am leading a cloning using pBluescript vector. When I arrived at the ligation step I realized that it involves specific working conditions applied in the case where cohesive ends are implicated which are different where blunt ends are present. I used the same enzyme Eco R V to cut both the plasmid and the DNA (insert).
EcoRV produces blunt ends on cutting.
https://www.neb.com/products/r0195-ecorv
If you go New England Biolabs (catalog or online) and look at the restriction enzyme you will see what ends are produced after the enzyme digest. The arrow heads above the recognition sequence indicate the "cut" site(s) in the sequence. This will help you determine which enzyme will result in blunt or sticky ends. (See attachment ).
Look on SmaI (blunt) and XmaI (sticky ends) both enzymes recognize the same sequence (cccggg).
also you can see the enzymes that produce the compatible cohesive ends. NEB catalog (at the end have appendix). look there when you have time. That is a nice summary and a lot of useul info such us: enzymes with compatible cohesive ends; cutting DNA fragment close to the end + other
you will benefit if you add more to your RG profile (like institution you work at; country and subject you work on.
You ask about designing the vector...>it depends on what you want to do with that vector. Tell me more what you want to do .WE have set of vectors in the lab if some is suitable > I will share with you
Info that you need to provide is: do you need that vector for cloning bacterial, plant or mammalian gene?
do you need to express/purify the protein? or other???
I added more informations to my RG Profile. I think that is better to change the vector. Thanks for your reply
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