Help! Making this construct is driving me crazy!

Amos H Heckendorf nestgrp at
Tue Jan 10 18:34:11 EST 1995

Carlisle Landel (landel at helios) wrote:

: We are trying to ligate a 5kb Not I fragment into a Not I digested
: plasmid that is about 13 kb in size.  The fragment is supposed to
: go into a Not site in the middle of a 10 kb piece of mouse DNA that
: is cloned into Bluescript II (from which the Not site has been 
: removed).  

: This should be straightforward, but it isn't working.  Are we up 
: against some sort of size limit?

The question I have is whether you are having trouble isolating the 
product.  We have heard that unless you are using NucleobondAX kits (we 
distribute them) you may see substantially reduced yields on the other 
kit.  If this is the case we have several solutions to help you with this.

: We've done controls that tell us that the bugs are competent, the ligase
: works, the CIP works, the restriction enzymes are cutting.

: We just aren't getting any inserts.

email me if you want the details. You may have a high gc content product 
that is reducing the yields as well.  We have ways to solve this problem too.

Amos Heckendorf (nestgrp at 800-347-6378 ; 508-481-6223
The Nest Group , Value Added Resellers for:
Macherey-Nagel, mfg of Nucleobond AX

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