Jump to content

  • Log in with Facebook Log in with Twitter Log In with Google      Sign In   
  • Create Account
  2. → Viewing Profile: Topics: Druss00

Druss00

Member Since 09 Aug 2012
Offline Last Active Jan 30 2013 02:21 PM
-----

Topics I've Started

Making DNA Linkers for cloning purposes

23 August 2012 - 08:58 AM

I want to know if anyone has tried this.  Design Oligos that will have 3' or 5' overhangs that correspond to a endonuclease restriction sites (I am trying to avoid cutting these linkers, so I thought I could engineer them already "cut").   I would then Ligate them using T4 ligase to phosphylate it.  Will this work?

Thanks!

Store western samples in NP-40 lysis buffer?

10 August 2012 - 09:09 PM

Hello,

I was just curious if I could store my western samples in NP-40 lysis buffer?  Usually I just add loading dye and Beta - Mercap and boil it, but I am going to be short on time tomorrow and was wondering if I could just freeze it down and add the reducing reagent later?

Thanks,
Wes

Need help for prepping western samples with NP-40

09 August 2012 - 09:01 PM

Hello,

I am new to western blots and have a question about prepping my tissue culture samples (293T cells).  After pelleting my cells and removing PBS (just to wash them) I add around 100uL of NP-40 to my pellet and re-suspend it.  After letting it sit on ice for 30 min (vortex every 10 min) I spin it down at the highest speed our centrifuge will go (this is all done in 1.6mL "epi" tubes) to pellet the nuclear DNA.  The problem is this.  When I take my tube out, I do see a nice white pellet of my nuclear DNA, but there is also some wispy gooey stuff at the surface of my supernatant.  I've tried to re-spin, but this stuff never pellets.  I don't want to continue my western with this stuff because I am sure it throws of my bradford measurements (to make sure I load equal amounts).

Any help would be appreciated.  Thank you!

Wes

  1. BioForum
  2. → Viewing Profile: Topics: Druss00
  3. Privacy Policy
Home - About - Terms of Service - Privacy - Contact Us

©1999-2012 Protocol Online, All rights reserved.