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
- - - - -

problem in cloning


  • Please log in to reply
2 replies to this topic

#1 gdbiomed

gdbiomed

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 25 April 2013 - 07:57 AM

I tried to over-express GST-tagged protein in PET41a(+) in BL-21 strain,but couldn't get the protein. The cloning and overexpression was done by other person -the gene is cloned in pGEMT VECTOR ,gel eluted in large scale and ligated to the PET41a(+), he got the sequencing results and overexpressed the protein, he didn't keep the plasmid alone instead in the BL-21 STRAIN ,THIS IS IN THE -80 FOR ABOUT 1 YEAR. I have used this for the overexpression -but didn't work to give protein. To ligate , i got the cloned pGEMTplasmid in DH5 alpha and did restriction -got the gene; but while doing PCR no amplification.please throw some light to get start with my doctorate ......,

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,676 posts
396
Excellent

Posted 25 April 2013 - 12:50 PM

Re-sequence the plasmid to make sure that the gene is there. Once that is done, assuming the plasmid is fine - try re-transforming the DNA into the BL21 and see how that goes. Some plasmids don't store well in the expression strains.

#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,454 posts
245
Excellent

Posted 25 April 2013 - 03:50 PM

I have heard (but not personally experienced) that there is a problem with storing BL21(DE3) strains that have a plasmid, and that you should retransform the strain rather than using a frozen stock. This never made sense to me, but perhaps it is true.




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

©1999-2013 Protocol Online, All rights reserved.