Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Low Miniprep Yield


  • Please log in to reply
1 reply to this topic

#1 TVo

TVo

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 01 August 2011 - 11:03 PM

Hey board,

I've recently been trying to miniprep pEntry vectors using home-made P1, P2, N3 buffers, filter/binding plates, etc. etc. (for the purpose of Gateway cloning). Previously, I've been able obtain quite a decent plasmid yield (~100 ng/uL). But it's been 7 months since my last miniprep spree of these vectors and, suddenly it seems, my yield has been very low (15-50 ng/uL). All of my buffers were freshly made except for P1 which was made ~7 months ago and did not have RNase A added until very recently (and stored at 4C). I tried to vary my DH5 alpha inoculation times between 16 and 28 hours with very little change in yield (previously when the yields were good, I inoculated anywhere from 18 to 20 hours). I always inoculate my bacteria in SOC media (should be richer in nutrients than LB) with kanamycin antibiotics.

Can it be possible that my P1 solution without RNase A and stored at 4C for 7 months got bad and somehow ruined the minipreps?


*Also, I do not reuse filter/binding plates or buffers. The buffers were made according to Qiagen's recipe or somewhere else I found. And I don't think it's due to variation in my pipetting...since I automate my minipreps. I just script something and a robot does it for me. =)

Edited by TVo, 01 August 2011 - 11:30 PM.


#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,589 posts
384
Excellent

Posted 02 August 2011 - 05:33 PM

It is possible that your P1 is off, though this is usually just salt, a buffer and some glucose. It may be that your NaOH has precipitated, if you have a stock solution, try preparing fresh and see how it goes.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.