Cloning and purity of DNA

Jianchi Chen JCHEN at MACC.WISC.EDU
Tue Jun 8 01:02:00 EST 1993


 
>After trying DNA fingerprinting on my Bumblebees without any variation
>at all (with 4 probes) I'm now planning to develop microsatellite markers
>instead. Since I have never cloned DNA I have some questions:
 
>1. How clean does my bumblebee DNA need to bee? In prior experiments I have
>   used Phenol/Chloroform extraction (one extraction with warm Phenol) and
>   then I have reprecipitated my DNA once. Is this enough?
 
Probably enough.  What you need is to do an enzyme digestion.  If the digestion
is complete, go ahead to do your cloning.
 
>2. I am planning to use Boehringers pUC Cloning kit. Comments?
 
pUC vectors such as pUC18/19 are pretty easy to use.  You don't even have to
buy a kit.  You can buy your vector and enzymes seperately and make up your
own buffer.  It works as well or maybe better.
 
>3. Do someone out there have any tips and hints that may be of use to me?
 
Since this is your first time, try the double enzyme (e.g. EcoR1 - HindIII)
cloning.  The effeciency is usually high and you don't have to use X-gal if you
don't like it.
 
Hope this can be a help.  Good luck.
 
Jianchi Chen
Food Research Institute
University of Wisconsin
Madison, WI 53706
jchen at macc.wisc.edu
 
 
>Thanks in advance.
 
>Peter Thoren
>Dept.of Genetics
>Uppsala University
>Box 7003; S-750 07 Uppsala; Sweden
>Phone: +46-18 67 13 23
>FAX:   +46-18 67 27 05
>E-mail: Peter.Thoren at genetik.uu.se
 
 
 



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