Restriction buffer composition (View forum version)



OA17

Posted 26 November 2009 - 08:34 AM

Hi!

I have to clone a fragment cut with SexAI from New England Biolabs, and RsrII (CpoI) from Amersham. The problem is that RsrII is a very old enzyme and I can not find the composition of the buffer in which it cuts, so I don't know if I can perform the double digest with both enzymes together or not.

Until this moment, I have made the digestions secuentially, using a millipore filter to dialize, but I would like to make them together in order to save time.

Does anybody know the composition of the RsrII (CpoI) buffer from Amersham (actually it is called buffer K), or can tell me where to find this information?

Thank you in advance!

klinmed

Posted 26 November 2009 - 12:48 PM

Hi!

I have to clone a fragment cut with SexAI from New England Biolabs, and RsrII (CpoI) from Amersham. The problem is that RsrII is a very old enzyme and I can not find the composition of the buffer in which it cuts, so I don't know if I can perform the double digest with both enzymes together or not.

Until this moment, I have made the digestions secuentially, using a millipore filter to dialize, but I would like to make them together in order to save time.

Does anybody know the composition of the RsrII (CpoI) buffer from Amersham (actually it is called buffer K), or can tell me where to find this information?

Thank you in advance!


x1 Amersham buffer K =20 mM Tris-HCl, pH 8.5, 10 mM MgCl2, 1 mM DTT, 100 mM KCl.

New England Biolabs recommend their buffer 4 + BSA for a double digest with SexA1 and RsrII.
I would use this rather than the Amersham buffer.

Hope this helps

OA17

Posted 27 November 2009 - 02:53 AM

Thank you very much!!

It helps a lot!

Have a nice weekend!