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
- - - - -

DNA cloning


  • Please log in to reply
4 replies to this topic

#1 feiliang

feiliang

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 09 May 2009 - 05:13 PM

Hi all;

I need your advice in DNA cloning.

How am I going to clone my DNA (include intronic site) for further protein expression study? My population study show 4bp deletion in intron4 of my gene is one of the significant risk factor for a genetic disease. I cant do cDNA cloning as it is in intron site. What should I do in order to characterize this deletion?

Thanks in advance.


Regards,
FL

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
406
Excellent

Posted 10 May 2009 - 04:48 PM

If it is a risk factor- is it just a statistical association? If so why do you need to clone it?

#3 feiliang

feiliang

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 11 May 2009 - 02:32 AM

I need to characterize it at RNA or protein level to see whether this deletion has anything to do with protein expression?

#4 micky74

micky74

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 11 May 2009 - 06:00 AM

I need to characterize it at RNA or protein level to see whether this deletion has anything to do with protein expression?



Right so:
to look for gene expression you need to run a Q-PCR in order to compare people bearing the insertion against people not bearing it.....so no need to clone it

Second problem if your mutation is altering a splicing site, I guess you need to clone your gene and see if it is spliced correctly or you are getting extra bands...look at the mini-gene or something over internet I guess there is a technique to look for aberrant splicing sites
If you have cells from people or your mutation is polymorphic in population you should consider an RNA extraction and amplification to look for different splicing in people with the insertion/deletion.

So cloning your gene doesn't make sense unless you know which technique you are going to use

#5 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
406
Excellent

Posted 11 May 2009 - 05:21 PM

Cloning an intron for expression studies also won't work, the constructs use bacterial expression style promoters etc, meaning that everything after the ATG is expressed, even when you transfect it into human cells.




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

©1999-2013 Protocol Online, All rights reserved.