PEG and Blunt end ligation

Charles A Miller oravaxcm at world.std.com
Thu Dec 14 17:42:02 EST 1995


I am currently trying to ligate a PCR product (~1000bp) into a vector by 
cutting the vector with SmaI to linearize it (leaving Blunt ends) and then
ligate it using the protocol given in Sambrook (Molecular Cloning). I have 
decided to do this since my PCR product has a SalI site on one end and an
EagI site on the other. While it is easy enough to cut the vector with SalI
and then EagI, the PCR products present a problem since SalI doesn't cut very 
well at all (the site is 3 bases from the end). So (as someone in this group
suggested), I am trying to first blunt end ligate it into one vector, grow it
up, then extract the plasmid and cut it out with SalI and EagI to put into 
the vector I chose. In the protocol given in Molecular Cloning, they suggest
using PEG or another chemical (didn't have it available) to increase the 
possibility of ligation. I am using PEG to accomplish this, but was wondering
whether it will adversely affect the cloning (i.e. do I need to clean the
plasmid first? What protocol would you suggest if this is so? I have a Wizard
PCR prep kit that I often use to clean CUT plasmid as well as PCR products 
which works fairly well, but does it clean ligated products?) If anyone has
any suggestions or protocols for this Blunt-end ligation and transformation
then I would be most grateful.

Thanks!

Chuck Miller
oravaxcm at world.std.com
 



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