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
- - - - -

question about transformation

Transformation of Pichia

  • Please log in to reply
1 reply to this topic

#1 jalapati

jalapati

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 20 August 2012 - 02:21 PM

Hi

Can any one please help me out with the transformation of Pichia pastoris?

We are trying to express gp96 (glyco protein 96) mutants in pichia pastoris.

I have tried electroporation as well as CaCl2 treatment for the production of competent cells.

But it was of no use Posted Image

After electroporation, I am adding 1mL of ice cold sterile 1M sorbitol and then incubating them for approximately 3 hours at 30o C and plating them on YPDS + Zeocin plates

I have also tried other methods like addition of 0.5mL sorbitol and 0.5mL of YPD media to the cells immediately after electroporation and then incubate them for 3 hours at 30o C.

Please send me your suggestions so that I can make it work.

Thank you.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,800 posts
406
Excellent

Posted 20 August 2012 - 02:39 PM

Check that your zeocin is the appropriate concentration - titrate the level needed for the cell strain you are working with.




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

©1999-2013 Protocol Online, All rights reserved.