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
- - - - -

PCR Sample Prep


  • Please log in to reply
4 replies to this topic

#1 jmerkin

jmerkin

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 14 July 2009 - 11:55 AM

I am looking to do RT-PCR from animal tissue. I would like to grind it in with a mortar and pestle in liquid nitrogen first to break it up. I am concerned about contamination if I work with multiple samples. I can use porcelain mortars, but they are porous and I am concerned about RNA getting up in there. I can use glass mortars, but I am concerned about them shattering when I put the liquid nitrogen in.

Does anybody have any experience or thoughts on this?

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,501 posts
252
Excellent

Posted 14 July 2009 - 05:21 PM

A bead beater with disposable tubes and beads might be a better choice of technique.

#3 ivanbio

ivanbio

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 116 posts
7
Neutral

Posted 15 July 2009 - 08:49 AM

I've used both successfully. In my experience the porcelain mortars work great for larger pieces of tissue (if you do not have much do not use these since you will lose a significant amount once your tissue is turned into a powder). Warning: make sure not to add more liquid nitrogen once your tissue has been turned into a powder and is dry. This may cause the powder to "puff" and you will lose part of your sample. Also work fast: once the tissue is a powder transfer it to the recipient you will use for extraction. This powder, when dry, will hydrate fast and make it harder to remove from the mortar. As for cross-contamination, I usually use multiple mortars at a time and once all have been used I stop to clean them all up before going through a new round of crushing.

Alternatively I suggest using a glass homogenizer. There is no need to use liquid nitrogen and they do a great job of homogenizing your sample.

Ivan
Carlsbad, CA

#4 Ytje

Ytje

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 23 July 2009 - 05:25 AM

I am looking to do RT-PCR from animal tissue. I would like to grind it in with a mortar and pestle in liquid nitrogen first to break it up. I am concerned about contamination if I work with multiple samples. I can use porcelain mortars, but they are porous and I am concerned about RNA getting up in there. I can use glass mortars, but I am concerned about them shattering when I put the liquid nitrogen in.

Does anybody have any experience or thoughts on this?


I work with gut tissue. First I cut the tissue up in a sterile petri dish with a sterile scalpel knife while the tissue is emerged in the lysis buffer (contains RNase inhibitors). After that I grind it (still in the buffer) in a tissue grinder (Potter-Elvehjem) at high speed. To finish it off I pass the lysate 10 times through a 21G neadle.
Works perfectly!

Ytje

#5 jmerkin

jmerkin

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 23 July 2009 - 05:56 AM

Thanks for the suggestions. I've gone the glass homogenizer route before, but I'm going to be dealing with quite a number of tissue samples, upwards of 80. The sheer (no homogenizer pun intended) number is what concerns me. I think I may go with chopping up and homogenizing.




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

©1999-2013 Protocol Online, All rights reserved.