phosphatase alkaline before cloning? - (Jun/03/2005 )

hi,

dear freinds I plan to do a manual protocol for cloning (no kit used). do you think is really necessary to treat the plasmid with Alkaline Phosphatase to remove 5´-phosphate groups from both ends of the plasmid (to ovoid self religation)?

it is possible to clone my insert even if I get very low copies of recombinant plasmids??
thanks in advance

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Hi,
If you are digesting your vector with two different restriction enzymes, then phosphatase treatment is not necessary at all. But make sure that your vectoe is cut completely by both the enzymes. If you are using onyl one RE better do the phosphatase treatment, and if u are using any phosphatase other than shrimp alkaline phosphatase , do an phenol/ethanol precipitaion after heat treatment to remove the phosphatase.
vinod

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Hi,

I would recommend to dephosphorylate your vector. I use enzymes and buffers
from New England Biolabs and I always do my dephosphorylations directly in
the same buffer after restriction, I just add 0.5 µl CIP per 20 µl (quite much I
have to admit) and incubate 1 h / 37°C. Then I remove uncut plasmid by
preparative gel electrophoresis and DNA-isolation from the gel.
Works very fine with low copy vectors up to 12 kb.

Regards,
Hennry

By the way: the two-enzyme-system described above only works for sticky ends, not for blunt ends. You have to keep that in mind.

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hi
this topic may helps.

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