3 replies to this topic

[sahil]

Dear all ,
I am just wondering how CIP treatment is done since I've never tried it before .Do we always need to purify the product after CIP treatment , what if the concentration of vector is too low .


Regards
Sandeep

[bob1]

sahil, on Jan 17 2010, 03:43 AM, said:

Dear all ,
I am just wondering how CIP treatment is done since I've never tried it before .

Add some CIAP to your reaction with the appropriate buffer, incubate for a while at 37 deg C.

Quote

Do we always need to purify the product after CIP treatment

Yes, otherwise it will remove phosphates from your other DNA (insert or plasmid) when you mix the two.

Quote

what if the concentration of vector is too low .

Make some more - you have glycerol stocks right?

[gauden]

bob1, on Jan 18 2010, 08:24 AM, said:

sahil, on Jan 17 2010, 03:43 AM, said:

Dear all ,
I am just wondering how CIP treatment is done since I've never tried it before .

Add some CIAP to your reaction with the appropriate buffer, incubate for a while at 37 deg C.

Quote

Do we always need to purify the product after CIP treatment

Yes, otherwise it will remove phosphates from your other DNA (insert or plasmid) when you mix the two.

Quote

what if the concentration of vector is too low .

Make some more - you have glycerol stocks right?

the same my situation. The concentration of vector ( M13) is very very low, it was un-visualized on agarose gel (0.8%) but there are a few because i try to transformant and get a few colonies. How can i resolve these problems. Many thanks for any suggestion

[leelee]

you could try Antarctic Phosphatase (http://www.neb.com/n...roductM0289.asp) which can be heat inactivated removing the need for purification- however I think you should go with bob's suggestion and just make some more- cheaper, and faster, to do it that way