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
- - - - -

Colony PCR for clone screen


  • Please log in to reply
4 replies to this topic

#1 zhgljj1998

zhgljj1998

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 18 January 2005 - 02:57 AM

Hi, is there anybody using Colony PCR to check your plasmid before sending sequence. is it necessary? thank you.

#2 leahf

leahf

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 45 posts
0
Neutral

Posted 18 January 2005 - 03:14 AM

Hi
not sure i understand your question...
before sending for sequencing it is usually advisable to check if your plasmid has the right-sized insert in it, and this can be done either by extracting the plasmid and cutting out the insert, or by PCR . Doing colony PCR is usually quickest and if you get a good unique band, then you can directly sequence the product without needing to extract plasmids at all.

in many cases it helps to dilute the colony in 50 - 100microliter ddw, and then take 1 microliter as template per reaction.

#3 Ali

Ali

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 19 January 2005 - 01:17 AM

I am not understand what do you mean?
If you want to check sequence of your recombinant plasmid (contain new insert), you should do colony PCR first.

#4 Great White Northern

Great White Northern

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 05 March 2005 - 09:09 AM

I have done it. It works well for a medium sized number of colonies to screen (say 5 to 20). For a lower number, I isolate plasmid and test via restriction analysis (hopefully, your insert contains at least one diagnostic restriction site) or nested primer analysis.
For over 20 colonies, I pick and patch, and screen via colony hybridization.
For colony PCR, you should be able to simply touch a sterile toothpick to your colony, and then dip it into your pcr solution. Always use a black as colony pcr is the only time I have ever had contamination.

#5 Scott

Scott

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 07 March 2005 - 09:39 AM

Hi,

I routinely do PCR colony screening, to determine positive clones, number of inserts and direction of inserts, especially if problems with background re-ligation. As far as is it necessary if you only have a few colonies with a single insert than just mini-prep, if it is anything more complex than use do colony screen.

Hope this has helped.

Cheers,

Scott




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

©1999-2013 Protocol Online, All rights reserved.