Sma I religation problem

Antonio Rodriguez Franco bb1rofra at seneca.uco.es
Mon Jul 17 12:29:43 EST 1995


In article <3tv50s$p5g at dingo.cc.uq.oz.au> mikep at biosci.uq.oz.au (Mike Poidinger) writes:
>From: mikep at biosci.uq.oz.au (Mike Poidinger)
>Subject: Sma I religation problem
>Date: Tue, 11 Jul 1995 23:27:02 GMT

>Hello everyone,

>I have been having major troubles with religating Sma I cut pGEM.  The vector
>has been cut, and the linear plasmid gel purified using Wizard clean up system,
>but try as I might it wont religate to itself.  The same routine performed on
>the same plasmid with Eco RI works just fine.

>Any help anyone has would be greatly appreciated.

>advTHANKSance,
>Mike

>------------------------------------------------------------------------------
>Dr Mike Poidinger      Now don't be lazy, 
>Microbiology, UQ       with the pleasure of sin  (Nitzer Ebb)
>Australia              
>mikep at biosci.uq.oz.au  
>-----------------------------------------------------------------------------

I do not know exactly what your problem is, but SmaI is a very tricky enzyme.
According the BioLabs brochure dealing with the M13 Cloning and Sequencing 
system, digestion of M13 with HincII and SmaI rise about 50 % colorless 
plaques when ligated in the abscence of experimental DNA. To fix this problem, 
they add, prior to adding T4 Dna ligase, 50 uM dNTP and 0,5-1 U of Klenow to 
the reaction, leaving the reaction to incubate for 15 min. Then you can 
proceed with the addition of your ligase.

This is true in our hands
Hoping this help



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