Molecular Genetics Questions!

[prenurse]

Ok anybody that can help me out with all this cutting/ligating stuff is greatly appreciated! I guess I need some help with either links/books, etc. Please explain! Thanks in advance!!

8. What new cleavage site(s) is (are) created when DNA is cut with HindIII, filled in by DNA polymerase I, and ligated?

9. A vector is cut with EcoRV and ligated to an insert cut with NruI. What new restriction enzyme recognition sequence is created?

10. A student is trying to cleave plasmid DNA grown in an unknown E. coli strain with AlwI. He knows two recognition sequences are present in the plasmid but the plasmid is not cut by the enzyme. Is the student using a “bad batch” of enzyme or is there another reason why the plasmid is not being cut? Suggest a solution to the student that would enable him to use AlwI to cut his plasmid.

[JackBean]

8. the recognized sequence is:
5'-A'AGCTT-3'
3'-TTCGA'A-5'

after cut:
A...AGCTT
TTCGA...A

after "filling"with DNA Pol
AAGCTT...AAGCTT
TTCGAA...TTCGAA

and ligate
AAGCTTAAGCTT
TTCGAATTCGAA

is there any new cut site?

9. EcoRV cuts GAT'ATC and NruI cuts TCG'CGA so, if you cut it and ligate back, what do you get? What RE cleaves in there?
http://www.neb.com/nebecomm/EnzymeFinde ... meIntl.asp
(you can search there also y sequence, that's useful for you;)

10. some REs are sensitive to presence/absence of some modifications, check whether this is the case