Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Cloning 4.2kb insert in 29.8 fosmid

Cloning 4.2kb insert in 29.8 fosmid - Protocols and Methods Forum

Cloning 4.2kb insert in 29.8 fosmid - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 12-01-2003, 04:09 AM
P.C.
Guest
 
Posts: n/a
Default Cloning 4.2kb insert in 29.8 fosmid



cloning can sometimes be a pain.

Deleted plasmids often result from re-ligation of your DNA in bacteria.
The fact that you see them by itself means that your cells are of quite
a good competency.

before you conclude that the insert is toxic:

1) check your ligation mixes on gel - if there was any degradation
(downward smearing) just re-prep your fragments.
2) do your ligations overnight, 16C (many say 1 hour "always" works,
however, longer ligation does always improve the odds).
3) check more colonies, of course. If you have a pair of primers (one in
the vector and one in the insert) it is easiest to check many by PCR
(directly from colonies).
4) Finally: avoid long UV exposure (!) with longer fragments it is even
more crucial. It can "kill" your DNA, and you even will not know it. If
you need more than 3 seconds to cut the band, it is too long (hand-held
low power UV lamp is the best). If you use a transilluminator for that
(it is a killer), be very carefull and illuminate only the part of the
gel that you are working with (one band at a time) and WORK FAST.

good luck,

Peter


[Only registered users see links. ] wrote:

Hi all,
It is weeks that I am trying to obtain a good contruction, I first did
ligations with these agarose
gel purified fragments, then I set up different ligation with different
molecular ratio 3:1, 1:1, 1:3
and I transfected E. Coli. The transfection was not very efficient but I
obtain several nice colonies
with all ratios and the control (cutted fosmid alone) was good (no
colonies).
But once I do minipreps and digestion controls, my construct is much
more shorter than what I
expect and not allways the same (8kb to 25kb). I think that there is
some deletions that occur
within bacteria and I tried different bacteria (DH5alpha heatshock, DH10
electro, XL1blue
heatshock) but I always have the same problem. Is my insert toxic? Do
you know some bacteria
strain wich is more appropriate fore such huge construct?
I would be very grateful if someone could give me some goog advices!
Kind regards.
Paul

Reply With Quote
Reply

Tags
298 , 42kb , cloning , fosmid , insert


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Cloning large insert in pQE30,expressing it! Chitra Molecular Cloning Forum 2 06-10-2010 09:38 PM
DNA Cloning Protocol moleculardude Molecular Biology Articles and Protocols 2 08-05-2007 02:31 PM
help on difficult cloning Senkovich, Olga Protocols and Methods Forum 1 05-05-2007 12:14 AM
Cloning Fails-- Trouble shoot please vijaykr Protocols and Methods Forum 7 11-09-2005 04:27 PM
Cloning 4.2kb insert in 29.8 fosmid onels@bluemail.ch Protocols and Methods Forum 0 11-28-2003 03:58 PM


All times are GMT. The time now is 12:01 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11666 seconds with 16 queries