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
- - - - -

purified dna using dh5alpha and qiaprep....plasmid not in genome


  • Please log in to reply
3 replies to this topic

#1 smhl12

smhl12

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 16 April 2010 - 12:33 PM

So I'm a chemist and new to the bio world. I just started using biotechniques to try to make some mutant proteins. I was given some transformed DH5alpha with ampicillin resistant plasmids and purified using qiaprep spin miniprep kit. I submitted the dna to the genome center and none of the samples contained any of the desired genes. However when i digested them and ran them through aragose gel comparing them to the plasmid that was used, the single and double digested strands matched up while the undigested strands did not. Anyone know what's going on? I am clueless

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 16 April 2010 - 01:25 PM

Undigested plasmid is circular, and can often be supercoiled (wound like a badly managed phone cord). These forms run peculiarly on a gel, often faster or slower than the corresponding linear fragment of the same size. Often, multiple forms are present, yielding several distinct bands, even though only one sequence is present.

#3 smhl12

smhl12

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 18 April 2010 - 02:55 PM

are you saying also that the plasmid was taken up, even though it is found nowhere in the genome?

#4 ProteinWork

ProteinWork

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 37 posts
0
Neutral

Posted 18 April 2010 - 03:27 PM

Your plasmid has ampicillin resistance gene and DH5alpha doesn't. So the fact that the cells can grow on selective medium containing ampicillin means that the plasmid has been taken up by the cells.

When you search your sequencing result for your target sequence, depending on the sequencing primer's position and direction, you might get reads on only partial sequence or the complementary sequence. Make sure that you don't miss it because of those. Use blast2n tool to compare your sequencing result to your target sequence.




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

©1999-2013 Protocol Online, All rights reserved.