Alk Phos treatment

ww40 William_D_WARREN at UMAIL.UMD.EDU
Wed Apr 22 12:47:00 EST 1992


>Hi There:
>
>A unique problem has surfaced in our lab and I was wondering if anyone else
has
>had a similar experience.  When using alkaline phosphatase to remove 5'
>phosphates from cloning vectors, has anyone ever had the problem of having the
>alkaline phosphatase treatment digest one's vector as well?  Various reagents
>have been changed, water, Tris, ZnCl2, the enzyme, all to no avail.  Does
>anyone have any suggestions or comments?
>

Yes...contaminating nucleases can be a real problem...

I get around it by phosphatasing at pH9 and with SDS in the
reaction - neither of which see to bother the phosphatase too
much but are not so pleasant for nucleases. For example, to a
20ul restriction digestion (containing say 1 ug of DNA) I add
1 ul of 10% SDS, 3.5 ul of Tris pH9 and the phosphatase -
incubate at 37 C for 30 minutes, add 1ul of 0.5M EDTA, extract
and precipitate.

Good Luck
BILL




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