morten.lukacs at biokjemi.uio.no (Morten Lukacs) wrote:
>I need some info. about Bacterial alkaline phophatase (BAP)
>>1).At which temperature is it recommened to incubate ??
>At 37oC or at maybe 60oC (to supress any exonucleases in the enzyme preparation)??
Follow the recommended protocol from the supplier, 37 degrees sound OK
(it's been awhile since I've used it. Don't overlaod the digest with DNA,
this will cause high background.
>2).Is if sufficient to purify the phosphatase treated DNA by simply load a sample
>on an agarosegel, and afterwards just purify the DNA from the gel....
>or is it necessary to do a phenol extraction and ethanol precipitation ???
This should be adequate to remove the enzyme from the prep. although it
implies that you will be treating a large sample. The a PVDF spin filters
available (Millipore) that will remove enzyme from the prep. in seconds
without the hassel of gel purification and increased UV exposure to you
and your sample.