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
- - - - -

RNA gel problem (melted bands)

RNA RNA electrophoresis RNA extraction

  • Please log in to reply
2 replies to this topic

#1 chlamychlamy

chlamychlamy

    member

  • Members
  • Pip
  • 2 posts
1
Neutral

Posted 27 January 2015 - 08:15 AM

Dear all,

 

I extracted RNA from Chlamydomonas cells with the traditional phenol:CHCl3 protocol (1X washing step) and precipitated it with LiCl (2.5 M final concentration).

Analysis at the Nanodrop gives good parameters and an amount of ~3 ug/uL (~3000 ng/uL), however when I loaded into the gel (5 uL + 1 stain), the gel after running (50 V, 50 min in TAE 0.5X) looks like the photo (MidoriGreen staining).

 

RNA degradation or some residual substances from extraction which interfere with running?

 

How is possible that the upper part of the run is without stain? Should I load less RNA?

 

Thanks!

Attached Thumbnails

  • RNAgel.jpg


#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,601 posts
265
Excellent

Posted 27 January 2015 - 11:16 AM

You will vastly overload the gel by loading 15 ug of RNA into a single lane. Dilute your sample 100x to 1000x and try again.



#3 chlamychlamy

chlamychlamy

    member

  • Members
  • Pip
  • 2 posts
1
Neutral

Posted 11 February 2015 - 05:34 AM

Thanks Phage! Yes, I overloaded the well, it's true..

But I had also a problem with TEA buffer because also DNA gels (right loaded) look completly wired.







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

©1999-2013 Protocol Online, All rights reserved.