Shrimp Alkaline Phosphatase

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Thu Oct 7 14:28:00 EST 1993


-----------------Klaus Writes--------------------------------
Hi Netters

We have recently started using USB Shrimp Alkaline Phosphatase for
dephosphorylation of our vectors.  

In the notes with the enzyme it states that heating at 65*C for 15 minutes
is sufficient to inactivate the enzyme.  Is this the experience of the net?
 If it doesn't inactivate of course then we will dephosphorylate our insert
also and get no transformants (which is our current result).

Any other comments?

****************????????????????*******************
Hi Klaus!
    I have used the USB's SAP enzyme and the CIP from
BM.  Both seem to work equally well.  There is one proviso;
I never heat inactivate any enzymes, always opting for the more 
effective phenol:chloroform extraction.
My logic is as follows;  What I may save by heat inactivating I
lose if the ligation doesn't work or if i don't get what I want.
I hate repeating ligations, so I take the extra 15 mins or so to phenol:
chloroform extract and ethanol precipitate. I know I don't need 
to but i still put the ethanol ppts in the -80  for 10 to 15 min  
and never have a problem.  Another trick is to keep MgCl2 to 10-20mM to 
enhance the precipitation and add Na acetate to 300mM.   This procedure
 always gives me good dephosphorylated vector DNA, unless the CIP is
 dead or dying, which has happened a number of times when the CIP gets
 about 6 months to a year old.  I think the key is if you are used to 
working with phenol and have a GOOD source of phenol that is not too expensive
then why take the chance!  I know i will probably get 100 people saying
that heat inactivation works every time for them.  Good keep it up! Just next
time your ligation fails to give you any colonies or gives a bunch of UCO's
(unidentified cloning objects) or gives no inserts consider your heat 
inactivation 
step.  Especially, as an earlier reply to your posting may have indicated,  
if you are doing something other than the very simple stick end ligation!
This is my $.02 worth.

Good luck with your ligations!  

Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________




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