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

DNA Extraction Forum DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


Miniprep problem

Miniprep problem - DNA Extraction Forum

Miniprep problem - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 04-05-2009, 10:19 AM
Pipette Filler
Points: 46, Level: 1 Points: 46, Level: 1 Points: 46, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2009
Location: Melbourne, Australia
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Miniprep problem



Hi all,

I performed a miniprep today (eager to get something I cloned off to sequencing).

Because I was so eager I thought, why not grow my XL1-Blue E.coli in YPTG media. I figured the more happy my cells are the more likely my yield of DNA will be greater. My vector of interest in this case is pET21-d (High or Low copy number, im not sure?....I should check). I did get alot of growth. So much so, that I decided to split the 10 mL overnights into two.

Anyway, I did the miniprep and speced the final product....890 ng/uL....1200 ng/uL. I know sounds dodgy right and there wasn't any bubbles. I was certainly hoping it was all my vector, and thinking that i'd grow these cells ON in YPTG again for miniprepping. However the better side of me thought to run a gel, and as suspected I got what looked like a whole lot of smearing from contaminating genomic DNA.

Another point I should mention is that during the miniprepping, the column seemed like it was clogged. I'm using promegas kit and I was extremely sure no precipitated material was put onto the column.

Any thoughts as too why im getting so much junk?
Can I use rich media to grow cells or is regular LB the only way to go?
Did i possibly have too many bacterial cells?
My cell lysis buffer (provided) had SDS crashed out of solution, so I heated it in a beaker of warm water. Could the warm buffer have anything to do with it?

Here's a pic of the gel
flickr.com/photos/37073882@N03/3413551499/
Reply With Quote
  #2  
Old 12-15-2009, 06:44 PM
Pipette Filler
Points: 155, Level: 3 Points: 155, Level: 3 Points: 155, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2009
Posts: 24
Thanks: 0
Thanked 3 Times in 2 Posts
Default Re: Miniprep problem

This has happened to me before. I think you are using higher than the recommended volume of bacteria for the miniprep spin columns. Try a switch to maxiprep if you can.
Reply With Quote
Reply

Tags
miniprep , problem


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
2D PAGE problem antheab Peptide Forum 3 12-26-2010 04:28 PM
SDS-PAGE problem Steve Nothwehr Protocols and Methods Forum 2 10-16-2009 08:59 AM
weird problem in transfecting 293 cells in 24-well plate Cell Biology and Cell Culture 1 04-17-2007 03:54 AM
N-body problem Torstein Raanes Physics Forum 1 12-01-2003 10:31 AM
Integration Problem / Finding Contour lines Problem blah12@mail.com Physics Forum 2 09-07-2003 11:28 PM


All times are GMT. The time now is 08:02 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 0.12012 seconds with 16 queries