stratagene pCR-script--- good or bad?

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Wed Apr 6 18:17:47 EST 1994


>Howdy folks, 
>
>Can any users of the above mentioned cloning vector/ kit post some
>opinions concerning performance?  I am currently cloning into pSP72 using
>the pharmacia sureclone kit and have found that it works fine but gives
>colonies without inserts a fair proportion of the time.  
>
>thanks,
>
>John Hess, PhD                    Phone me 916 752 8420
>Dept of Human Anatomy             FAX me 916 752 8520
>University of Calif               Email me jfhess at ucdavis.edu
>Davis, CA                         or leave me alone, your choice.


The kit does work but the enzyme is VERY expensive.  You need blue/white
selection because almost half of the colonies are religation.  We have
engineered pGem7 with an Srf1 site and buy the enzyme in (~$A800.00 for
300U from Stratagene). (Other sources of Srf1??? anyone??)  

If your insert does not have a Sma site use Sma instead.  This also works.

Hints
1. DO NOT ligate for longer than an hour or so at 25*C or your background
increases.  
2. Use Sma to cut your vector and then use the Srf1 in the ligation.  Since
Sma is FAR cheaper.

Cheers, Klaus

Disclaimer: not all this info is from my lab but the other sources should
be reliable. K
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
PO Box 334, Canberra, ACT 2600, Australia
E-mail: Klaus.Matthaei at anu.edu.au

"I'd rather a full bottle in front of me than a full frontal lobotomy"
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