Cloning and purity of DNA
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.
Food Research Institute
University of Wisconsin
Madison, WI 53706
jchen at macc.wisc.edu
>Thanks in advance.
>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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