A GOOD KIT FOR PCR CLONING
jarrell at smaug.ucr.edu
Thu Jan 26 19:43:42 EST 1995
I am using the pCR-Script kit from stratagene. It utilizes the rare cutting SrfI
enzyme in the ligation mix to minimize vector self ligation (the vector forms an
SrfI site upon self-ligation and is cut). None of the genes I have cloned to
date have contained sites for this enzyme but it is worthwhile checking if your
cloning attempt is not successful.
I have been very happy with the
results. The manual is very thorough with several optional steps that I use
(blunting the PCR products with Pfu polymerase). I am getting lots of white colonies
and the false positive rate is very low. The controls are easy to implement also.
You don't need a different antibiotic for the basic transformation control as in the
TA Cloning kit from InVitrogen. If you feel the need to check the false positive rate you do have to make a LB/chloramphenicol resistance (all other
plates are LB/Amp/Methicillin.
Everyone else in my lab uses the TA cloning kit and seems happy with it. I started
using it when it was first marketed and developed a strong distrust of it. The vector
had some bacterial insert that was either a contaminant or not removed in the
processing. I also find the manual to be too brief for my taste, but I'm
a detail hog.
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