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

Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


A mystery!!! Plasmids smear when digested with site-specific enzymes

A mystery!!! Plasmids smear when digested with site-specific enzymes - Molecular Cloning Forum

A mystery!!! Plasmids smear when digested with site-specific enzymes - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-07-2010, 09:06 PM
Pipette Filler
Points: 18, Level: 1 Points: 18, Level: 1 Points: 18, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2010
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Question A mystery!!! Plasmids smear when digested with site-specific enzymes



This is my first time using this forum and also my first post. I'm hoping that someone out there can help me out and explain why I'm seeing what I'm seeing.

For my cloning, I have been using PMC103, a e.coli strain that is deficient in recombination. For the past couple of years, I have had no problems with this strain and was able to clone and also reclone vectors from this strain. However, lately, I have been noticing that whenever I transfect plasmids (not just ligation reactions) into this strain, grow them up and miniprep the plasmid derived from this new strain, the plasmid smears whenever I tried to digest them with site-specific enzymes. (I didn't over-digest and checked for star activity) I have done this with several types of plasmids, different combinations of enzymes and have also sequenced the plasmid to make sure that the plasmid did not recombinate. Even though the sequence data is good, plasmids derived from this strain always produces a smear. I've also made newly competent cells for this strain, and the same smearing phenomenon is produced.

The really interesting thing is that when I take plasmids (the ones that always smears) from the PMC103 strain and then transfect them into a different strain, such as STBL4, the plasmid is normally digested and does not smear. Can someone explain this to me?

Last edited by doug1c; 10-07-2010 at 09:10 PM.
Reply With Quote
  #2  
Old 10-09-2010, 01:25 AM
jlf jlf is offline
Pipette Filler
Points: 380, Level: 7 Points: 380, Level: 7 Points: 380, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 23
Thanks: 0
Thanked 6 Times in 6 Posts
Default Re: A mystery!!! Plasmids smear when digested with site-specific enzymes

Did you miniprep with too many cells? If you use too many cells, the lysate will be very viscous and a lot of the chromosomal DNA will shear and contaminate you plasmid. When you digest with restriction enzymes, chromosomal DNA will cleave at different lengths and manifest as a smear.
Reply With Quote
  #3  
Old 10-29-2010, 01:11 AM
Pipette Filler
Points: 6, Level: 1 Points: 6, Level: 1 Points: 6, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2010
Posts: 4
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: A mystery!!! Plasmids smear when digested with site-specific enzymes

I had this same problem with Stbl3. Stbl3 and PMC103 are not endonuclease deficient (endA-). You end up with a bit of endonuclease activity in your plasmid, then when you incubate at 37 degrees for your restriction digest, your plasmid gets chopped up in random places as well. The solution is simple: you must do the "optional" wash step in your miniprep kit that comes before the {70% ethanol + salts} wash. For example, in the Qiagen miniprep kit, you must do the 0.5 ml PB wash before the 0.75 ml PE wash. If that still doesn't work, try heating the PB to 50 degrees or doing two PB washes. The chaotropic salts in PB (or equivalent) will denature the residual endonucleases.
Reply With Quote
The Following User Says Thank You to mutantmutant For This Useful Post:
danfive (10-29-2010)
Reply

Tags
cloning , digest , digested , enzymes , mystery , plasmids , pmc103 , sitespecific , smear


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


All times are GMT. The time now is 08:50 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12473 seconds with 15 queries