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.


problems with restriction digestion

problems with restriction digestion - Protocols and Methods Forum

problems with restriction digestion - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 05-11-2009, 07:50 PM
Matthew Martino
Guest
 
Posts: n/a
Default problems with restriction digestion



I agree. There is always the concern of Methylation, a cells natural defense of dna from restriction enzymes. If the extra bands appear and they seem faint, for instance dimmer than the band above and the band below it, then you are having partials. This can easily be explained by the fact that some enzymes require exact salt conditions for activity and anything differing from the optimum will result in partial digests. One way to check if this is the case is doing digests with the enzymes individually. You shouldsee linear DNA fragments in both cases (slight smiling of bands, but no streaking and definitely not multiple bands such as linear and supercoiled running together). Whichever enzymes doesn't give you completely linear DNA is the problem.

Matt
Reply With Quote
  #2  
Old 09-16-2011, 06:22 PM
Pipette Filler
Points: 811, Level: 16 Points: 811, Level: 16 Points: 811, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2011
Posts: 2
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: problems with restriction digestion

Hello,
I need help trying to figure out why my restriction digestion is not working. Here is how I did it.
I cultured bacteria that were already transformed with the my gene of interest using pcDNA3. I purified plasmid DNA from the culture using GenElute Five-Minute Plasmid Miniprep kit. Then measured DNA concentration in sample and the 260/280 value was 2.1.
Then by PCR, I amplified the gene and then ran the product on a gel to confirm that the correct product was amplified. I used GenElute PCR cleanup kit to purify the PCR product and then cloned the gene using pLenti6/V5 Directional TOPO cloning kit.

Then purified the plasmid DNA using GenElute Five-Minute Plasmid Miniprep kit. After that I incubated 1 ul (containing 5 ng) of DNA with 1 ul BamH1 (50U/uL) for 1 hr at 37 degree Celcius in a dry incubator. On running the restriction digest on a 1% agarose gel, I see no bands in the digest.

I do not know where I am going wrong. I see the plasmid band when I run the undigested DNA on a gel.

Suggestions will be really helpful.
Reply With Quote
  #3  
Old 12-16-2011, 10:16 AM
Pipette Filler
Points: 39, Level: 1 Points: 39, Level: 1 Points: 39, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2011
Posts: 5
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: problems with restriction digestion

Really this is a big problem.so is there any solution for that?
Reply With Quote
  #4  
Old 01-04-2012, 12:40 PM
Banned
Points: 107, Level: 2 Points: 107, Level: 2 Points: 107, Level: 2
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2012
Posts: 13
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: problems with restriction digestion

i agree that this is a big prob ...any solution ?
Reply With Quote
Reply

Tags
digestion , problems , restriction


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
problems with restriction digestion chemophoto@gmail.com Protocols and Methods Forum 7 01-04-2012 01:00 PM
doubt regarding agrobacterium DNA restriction digestion Michael Sullivan Protocols and Methods Forum 1 05-06-2011 08:57 AM
Restriction Enzyme Digestion Protocol admin DNA Techniques 1 04-13-2011 06:11 AM
Restriction digestion admin Article Discussion 0 03-16-2007 05:34 AM
Restriction Enzyme Digestion admin DNA Techniques 0 08-02-2006 09:44 AM


All times are GMT. The time now is 04:08 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 0.12996 seconds with 16 queries