NdeI: cutting problems?? anyone else?? seems silly! (fwd)

Jody K. Hirsh jkh141 at nwu.edu
Tue Apr 16 13:12:14 EST 1996

In article <4kl3n8$5p4 at africa.cis.co.za>, PRETOR at PINELN.AECI.CO.ZA 
>In article <dmorris-1503961110250001 at thermo.sbs.auckland.ac.nz>, 
dmorris at sbsnov1.auckland.ac.nz (daniel morris) says:
>>Ditto.  Use lots of enzyme, overnight incubations preferably, add more 
>>good luck every now and then, use CLEAN DNA, and allow at least six
>>base-pairs of extra sequence when designing primers containing 
>>NdeI sites.
>>Its a bastard of an enzyme.
>It also helps if one increases the digestion volume.
I unfortunately missed the original post.  However, if you are having trouble 
cutting PCR products after  amplification and don't want to buy new primers 
 with more bases on them, then you can use a Methods in Enzymol. 
method--ligate the PCR products together and then digest with the 
restriction enzymes and then subclone the products.  This will also work 
with 1/2 restriction sites of 6-mer palindromic sites.(V. Jung et al.,Meth. in 
Enzymol.  218:357-62, 1993).
Jody K. Hirsh
Northwestern University, Chicago, IL.   USA
jkh141 at nwu.edu

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