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.


urgent help please

urgent help please - Protocols and Methods Forum

urgent help please - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 05-29-2006, 01:12 AM
Jayakumar, R
Guest
 
Posts: n/a
Default urgent help please



I U degrades approximates 1 ug of DNA in 10 min at 37C in appropriate
reaction buffer. Your buffer does not seem to be appropriate since it
has EDTA which is an efficient chelator and will inhibit DNase activity.
It would work well with a sonicator.
Try to use a buffer without EDTA but containing other protease
inhibitors (Roche complete protease inhibitor cocktail is a good
alternative (the one without EDTA)). For optimum activity, DNase
required Mg++ in buffer.
Try out this protocol
[Only registered users see links. ]
ase/Purification/ec_Ecoli_enzyme.html

Best of luck
Jai



-----Original Message-----
From: [Only registered users see links. ]
[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of chiranjit
chowdhury
Sent: Sunday, May 28, 2006 1:26 PM
To: [Only registered users see links. ]
Subject: urgent help please

Hi!!

I am a research scholar of dept of Biotechnology ,IIT
Kharagpur,India.Myexperiment simply involves growing E coli containing
a pPBP fusion
construct,lysing the cell,purify the protein.Now I don't have access to
a
sonicator.So I simply followed the protocol recommended by New England
Biolabs and prepared a lysis buffer containing lysozyme,EDTA,Tris and
NaCl
(pH8).The volume of this buffer is 50 ml.But the main problem I have
faced
is the suspension becomes highly viscous due to chromosomal DNA and it
becomes very hard to be taken out.I know that DNase could solve the
problem
but I don't know how much should I have to add. My DNase I (RNase
free)conc
is 1U/ul.

With worm regards..



chiranjit chowdhury

research scholar

iit kharagpur

India
_______________________________________________
Methods mailing list
[Only registered users see links. ]
[Only registered users see links. ]


This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you.

Reply With Quote
  #2  
Old 05-29-2006, 02:13 PM
Wolfgang Schechinger
Guest
 
Posts: n/a
Default urgent help please

in that case, (as a means of emergency) increase the magnesium
concentration to exceed the chelating capacity of EDTA

Wo

Reply With Quote
  #3  
Old 05-30-2006, 12:43 PM
Louis Hom
Guest
 
Posts: n/a
Default urgent help please


And actually, having 10 mM MgCl2 on its own (in the absence of EDTA of
course) will make the DNA quite easy to pellet so that you can just spin
it out instead of shearing it or digesting it. However, I strongly
recommend adding it at the same time as the lysozyme, NOT after the cells
are already lysed. Granted, it makes it harder to tell when the cells
have lysed, but it works really well for me.
--
__________________________________________________ ____________________________
Lou Hom >K'93
[Only registered users see links. ]
[Only registered users see links. ]
Reply With Quote
Reply

Tags
urgent


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
urgent help in this issue adeltaher Stem Cell Forum 1 01-20-2009 08:45 AM
Urgent issue!!!!: gene ID conversion tools Hatam BioStatistics Forum 0 07-11-2007 02:13 AM
urgent ...pls help julie.white1@gmail.com Botany Forum 1 07-07-2006 03:43 AM
Urgent request: BME required for RNA isolation? Deanne Bell Protocols and Methods Forum 2 02-07-2005 10:38 PM
Urgent request: BME required for RNA isolation? Deanne Bell Protocols and Methods Forum 0 02-07-2005 06:55 PM


All times are GMT. The time now is 05:30 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 0.13270 seconds with 16 queries