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how CIP treatment is done


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#1 sahil

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Posted 17 January 2010 - 02:43 AM

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 .


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Sandeep

#2 bob1

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Posted 17 January 2010 - 03:24 PM

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.

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.

what if the concentration of vector is too low .

Make some more - you have glycerol stocks right?

#3 gauden

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Posted 20 January 2010 - 09:16 PM

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.

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.

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

#4 leelee

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Posted 20 January 2010 - 09:37 PM

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 :)




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