Yi Zhang (BIO) yzhang1 at
Thu Nov 13 16:25:37 EST 1997

Hi Nico:

It sounds so familiar, as a matter of fact, two persons in our lab are
still trying to figure our what went wrong with their cloning. They all
used the Qiagen kit (gel extraction and miniprep) and have the similar
problem. I did miniprep with Qiagen kit (miniprep kit) recently and did
not get enough DNA (and not pure enough for sequencing). I then used phage
to get single stranded DNA and sequencing worked. I think your problem may
be due to the kit. I usually donot use Qiagen kit for gel extraction. I
use Bio101 all the time and works everytime so far. Another thing is the
de-phosphorylation step which could be trouble-some even you checked the
fragment on the gel. I had some problem with that before. I recently clone
a construct using the massy way, digestion and gel extraction, and
cloning. Only have to screen more colonies. I have got my clone from
screening 20 colonies. (1/20). On the other hand, you can do colony
hybridization to screen more and save you a lot of time. Hope this will
help and maybe use different method if you consistently get the same
problem. Good luck


						* ___ 		
Yi (Ian) ZHANG,					*   | | /	
IP2CMB, IBS					* --- |/		
USF, Tampa, FL					* |__----
Web site:*   | |\	
						*   | |/\
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On 13 Nov 1997, Nico Dantuma wrote:

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