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
- - - - -

pcDNA 3.1 Directional TOPO clone issues

ligation transformation

  • Please log in to reply
18 replies to this topic

#16 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,674 posts
396
Excellent

Posted 14 December 2013 - 01:20 AM

So what;s the problem if you have correct clones - most of the time you only need one, which you would then grow up and prepare glycerol stocks of, thereby creating a more or less continuous supply of plasmid.



#17 Jeremy55

Jeremy55

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 14 December 2013 - 01:29 AM

So what;s the problem if you have correct clones - most of the time you only need one, which you would then grow up and prepare glycerol stocks of, thereby creating a more or less continuous supply of plasmid.

I'm gonna construct one hundred clones of a virus protein from different subtypes. That's why I need a good success rate, or the cost would be great.



#18 Jeremy55

Jeremy55

    member

  • Active Members
  • Pip
  • 14 posts
0
Neutral

Posted 20 December 2013 - 03:22 AM

Hello, everyone

I'm fresh here. And I have some problems with pcDNA 3.1 Directional TOPO cloning. I've been trying to insert a 3KB fragment into the pcDNA 3.1 D/V5-His-TOPO vector,then transform the recombinant plasmid into TOP10 competent cells. However, there are no or very few colonies on the plate.

I need all of you experts' instructions. Help me please.

 

Well,I have solved the problem of using pcDNA 3.1 Directional TOPO vector.And I will post my solutions for this  problem when I have free time .In the end, I wanna say thank you to bob 1, a very kind and enthusiastic guy. Good luck to you all.



#19 Muzamil

Muzamil

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 29 June 2014 - 04:21 AM

Hi Jeremy,

I'm facing a same problem as you had, with the cloning of 3kbp insert into pcDNA3.1 D TOPO vector. I'm getting only false positive colonies.

Now that you have overcome this problem, it would be grateful on your part if you kindly tell me how it worked.

 

Regards







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

©1999-2013 Protocol Online, All rights reserved.