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
- - - - -

Does a single colony of E.coli contain more than one plasmid


  • Please log in to reply
7 replies to this topic

#1 resmi

resmi

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 27 October 2010 - 08:53 PM

HI,

I am working on pHELLSGATE12 vector for RNAi in plants. Initially I clone my gene in pDONR221 vector and LR recombined into pHELLSGATE12 vector. I got numerous colonies on spectinomycin plate which on colony PCR and sequencing confirms the presence of insert (Using gene specific primer I got gene preceeding some regions of (approximately 300 bp) pDONR221?????). But on sequencing with pHELLSGATE specific primer (CaMV and OCS) I got approximately 800 bp reading in both orientation which is of the pEHLLSGATE vector region only. So whats happening. I was confused???????? Does anybody know the solution......... If a single colony contain more than one plasmid how should I proceed...

#2 Ameya P

Ameya P

    Rervm Cognoscere Cavsas

  • Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 330 posts
25
Excellent

Posted 27 October 2010 - 09:02 PM

But on sequencing with pHELLSGATE specific primer (CaMV and OCS) I got approximately 800 bp reading in both orientation which is of the pEHLLSGATE vector region only. So whats happening. I was confused???????? Does anybody know the solution......... If a single colony contain more than one plasmid how should I proceed...


It would be nice if you could confirm that the sequencing primer site was close to the actual insert (less than 800bp). As far as I know, sequencing reads are usually 800 bp and if you primer site is too far, you will just get the vector sequence and no insert sequence (even though the insert is present). I did not quite understand your 'more than one plasmid question'. Do you think that you have a colony with a realigned vector and a vector with the insert???? Kindly clarify.

NEW!!!!  5 Reasons why Rosetta is a SUPERSTAR on CoffeeTableScience!!!!

Image copyright: Adrian Koh SF.
Replication of this art is strictly prohibited without express permission of the artist


#3 resmi

resmi

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 29 October 2010 - 01:16 AM

I had done the gateway cloning using entry and destination vector.And after LR recombination i had sequenced the transformed clone (????)with the primer which should read the insert. But I couldn't get the read (insert region) even after 750 bp (only the vector region). But there is insert in the colony which I confirmed by gene specific primer which is an enigma for me. Then why should I doesn't got the insert using vector specific primer (primer very near the recombination site).

#4 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 29 October 2010 - 03:36 AM

If you're asking whether a single cell of E. coli can contain two different clones based on the same vector -- one clone with an insert and one without, in this case -- the answer is no. Since the vectors are the same, they belong to the same incompatibility group, and the cell cannot maintain more than one clone. There will, of course, be more than one copy of the unique clone in the cell; how many copies exist in a single cell is determined by the vector's copy number.

#5 resmi

resmi

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 01 November 2010 - 12:23 AM

If you're asking whether a single cell of E. coli can contain two different clones based on the same vector -- one clone with an insert and one without, in this case -- the answer is no. Since the vectors are the same, they belong to the same incompatibility group, and the cell cannot maintain more than one clone. There will, of course, be more than one copy of the unique clone in the cell; how many copies exist in a single cell is determined by the vector's copy number.



#6 resmi

resmi

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 01 November 2010 - 12:25 AM

If I inoculate from an E.coli colony and isolate plasmid then is there any chance that i got vector alone and insert relying on another vector?????????

#7 pDNA

pDNA

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 493 posts
14
Good

Posted 01 November 2010 - 03:50 AM

If you inoculate your colony PCR from the original ligation plate there is the possibility that you get a false negative signal due to traces of the original vector that was spread with the ligation reaction. You can streak-out your clones on a fresh plate and try to screen again ...you will see that this false negativ signal will go away!

Regards,
p

#8 Ragu

Ragu

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 16 December 2010 - 11:53 PM

Hi Resmi,
Did you find the answer?? I have a question.
Is your HG12 is called pStargate?? If so there might be a problem with your construct.
Try to match/blast the sequence result against your vector sequence and identify where it is amplifying. You will get an idea.
In my experience, the CSIRO RNAi vectors has some kind of problems like intron reversal etc.




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

©1999-2013 Protocol Online, All rights reserved.