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
- - - - -

How to know the exact plasmid size?


  • Please log in to reply
6 replies to this topic

#1 tyrael

tyrael

    member

  • Active Members
  • Pip
  • 23 posts
0
Neutral

Posted 27 March 2009 - 06:31 PM

Hi all.

i m now working onto some new isolated bacteria on my own and i have no idea that wat kind of plasmid in inside them.
i have extracted out the plasmid DNA and did a gel run.

and it show me with some multiple bands, yes, i know it's common for plasmid DNA due to its conformation (supercoiled, nicked and linear), so it gives me different migration pattern.

my question is,

how do i determine which is the EXACT size of the plasmid that i extracted ? since it migrates differently though at same size ?


pls correct me if i am wrong.
thank you.

#2 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 27 March 2009 - 06:41 PM

The only way to know the EXACT size is to sequence it. You can get an approximate size by cutting it with a restriction enzyme and seeing what size fragments are produced. It might take testing with a few different enzymes to find one that cuts into only a few fragments, so you can add the fragments sizes up to get the total.

#3 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,113 posts
59
Excellent

Posted 27 March 2009 - 09:25 PM

yes as HomeBrew said you need to cut your plasmid to give you a linear plasmid then run on the gel again. but my question is that if your bacteria strain is already known, then can't you find that plasmid sequence at Genebank or somewhere to figure out what RE would give you only 1 linear band?

#4 T C

T C

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 277 posts
0
Neutral

Posted 27 March 2009 - 10:19 PM

Hey,

Run standard vectors without digesting and compare their mobility and estimate aproximate size of yr plasmid.

Hope it helps. :rolleyes:

Best,
TC

#5 tyrael

tyrael

    member

  • Active Members
  • Pip
  • 23 posts
0
Neutral

Posted 27 March 2009 - 11:35 PM

but my question is that if your bacteria strain is already known, then can't you find that plasmid sequence at Genebank or somewhere to figure out what RE would give you only 1 linear band?


but different bacteria may possess different plasmid one, right ? then how could i be so sure that which plasmid that it have ? :rolleyes:

#6 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 28 March 2009 - 02:56 AM

I agree with TC. Cut the plasmid with a restriction enzyme. And run the DNA fragments with a DNA ladder.

You might get several DNA fragments from the digest. Just sum the sizes of the DNA fragments.

Repeat this for several different enzymes just in case an enzyme does not cut or cuts too frequently.
May your PCR products be long, your protocols short and your boss on holiday

#7 tyrael

tyrael

    member

  • Active Members
  • Pip
  • 23 posts
0
Neutral

Posted 28 March 2009 - 03:10 AM

:lol: Ok. Thank you all so much! i will proceed with the restriction then. =)




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

©1999-2013 Protocol Online, All rights reserved.