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
- - - - -

E Coli strain for lentiviral packing vector


  • Please log in to reply
4 replies to this topic

#1 PostDocTrauma

PostDocTrauma

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 37 posts
1
Neutral

Posted 17 July 2011 - 01:03 AM

Hi all,

I've got some lenti vectors but I can't get the packaging vector (delta R) to transform into my dh5alpha. I only bought the cheapest subcloning efficiency ones (they worked for all the others, including the supposedly toxic insert....

I've tried transforming 2ng, 5ng and 10ng but nothing. I am going to plate the rest of the bacteria but I suspect there won't be much there.

We have some TOP10 in the lab, but can anyone recommend another strain to transform something over 10kb?

Thanks :)

#2 pDNA

pDNA

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 493 posts
14
Good

Posted 17 July 2011 - 01:45 PM

you should use a E. coli strain that provides high transformation efficiency and extremely low rates of recombination ...for retroviral stuff people often use STBL2,3 or 4 cells ...as far as i remember the STBL4 are said to be good for large constructs (eg. cosmids).

Maybe you should also increase the amout of DNA you use when working with large plasmids.

Regards,
p

#3 PostDocTrauma

PostDocTrauma

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 37 posts
1
Neutral

Posted 19 July 2011 - 11:50 PM

I tried to transform with 40ng for both dh5alpha and top10. both didn't work. I can't really afford to spend any money for the next few weeks as it's the end of the financial year.

Guess I will have to buy the STBL lines then

#4 pDNA

pDNA

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 493 posts
14
Good

Posted 20 July 2011 - 02:15 AM

you can also check the quality of your plasmid on an agarose gel ...maybe it is already degraded or broken by repeated freeze-thaw cycles.

Regards,
p

#5 PostDocTrauma

PostDocTrauma

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 37 posts
1
Neutral

Posted 20 July 2011 - 05:11 AM

I don't have much left so whatever it is will have to do :(

we don't use ethidium so will have to run at least 50ng.




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

©1999-2013 Protocol Online, All rights reserved.