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
- - - - -

Electroporation strange problem


  • Please log in to reply
1 reply to this topic

#1 biolab12

biolab12

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 23 December 2009 - 05:35 AM

Hi all,

I am trying to transform electrocompetent DH5 alpha cells with a standard plasmid . I use 40 ul of the cells and about 50ng DNA. Electroporator is from eppendorf. Conditions : 1450 V using 0.1 cm cuvettes (time constant I get is 5.4 ms ). After electroporation, I let the cells recover in LB for about 1 hr at 37 deg.

I normally centrifuge the sample and remove about 800ul LB and resuspend the cells in remaining 200ul and plate it on appropriate LB plates.

I have never had problem with this technique and always had high transformation efficiency. But recently with the new batch of electro competent cells, I see a strange white jelly like thing after I centrifuge my cells .I also have no colonies after transformation. I am guessing the cells are dead and perhaps it is the cell debris that I see after centrifugation. Is it because of the pulse from my electroporator?

Anybody had a similar problem or any suggestion?

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,499 posts
252
Excellent

Posted 23 December 2009 - 05:52 AM

Possibly, or the cells could be dead prior to electroporation. You can test this by serial dilution of the cells and plating on non-selective medium. Your electroporation voltage sounds high. I would normally expect around 1.1 to 1.3 KV for a 1 mm cuvette. I usually do recovery in SOB not LB, but this is almost certainly not your problem. I'd suggest just making new electrocompetent cells.




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

©1999-2013 Protocol Online, All rights reserved.