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
- - - - -

very high molecular weight plasmid


  • Please log in to reply
6 replies to this topic

#1 soymilk14

soymilk14

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 81 posts
1
Neutral

Posted 28 April 2010 - 05:57 AM

hi everyone. i recently prepared maxiprep of a plasmid with gfp. this plasmid was commercially available and just transformed them and prepared maxiprep. the plasmid i purified has an OD of 1.9 so i think its okay however when i checked it in agarose, the size is very high, higher than the first line of the dna marker. I expect that the MW of my plasmid would be around 5-6Kb only.

#2 Clare

Clare

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 192 posts
1
Neutral

Posted 28 April 2010 - 06:46 AM

Did you digest your vector before running it on a gel?

Non-linearised DNA will run slower than linear DNA and hence look bigger in size compared to a DNA ladder.

Clare

hi everyone. i recently prepared maxiprep of a plasmid with gfp. this plasmid was commercially available and just transformed them and prepared maxiprep. the plasmid i purified has an OD of 1.9 so i think its okay however when i checked it in agarose, the size is very high, higher than the first line of the dna marker. I expect that the MW of my plasmid would be around 5-6Kb only.



#3 soymilk14

soymilk14

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 81 posts
1
Neutral

Posted 28 April 2010 - 07:07 AM

hi clare, no, i did not digest it. i can try digesting it then clare and check. it is a plasmid bought commercially, harboring a gene fused with gfp. it was running out so i transformed it and prepared maxi prep. i just dont know why after running its molecular weight is very high. could it be chromosomal dna?

#4 epibio

epibio

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 33 posts
0
Neutral

Posted 28 April 2010 - 08:11 AM

How did you purify the plasmid? If the DNA is running near the wells, it could be chromosomal DNA contamination.

#5 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 28 April 2010 - 09:03 AM

You can not get an estimate of a circular DNA's size by comparing it to a linear DNA ladder...

#6 soymilk14

soymilk14

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 81 posts
1
Neutral

Posted 28 April 2010 - 05:02 PM

You can not get an estimate of a circular DNA's size by comparing it to a linear DNA ladder...



oh thank you thank you. i purified it by etbr-phenol-chloroform after maxiprep. are there any suggestion how to confirm if my plasmid i run on the gel and purified is correct. sequencing?

#7 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 28 April 2010 - 05:06 PM

Sequencing is one way. Another less exact method is to digest the plasmid with a restriction enzyme that linearizes it, and then you *can* get an estimate of its size compared to your ladder.




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

©1999-2013 Protocol Online, All rights reserved.