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
- - - - -

annealing oligos


  • Please log in to reply
3 replies to this topic

#1 lyok

lyok

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 206 posts
6
Neutral

Posted 29 July 2014 - 10:17 AM

Hallo all,

 

I have some oligos to anneal to clone into a plasmid.

 

The problem is however that one of the people that also uses these dissolved them in the wrong buffer.

 

We normally use an annealing buffer of 1mM EDTA, 50mM NaCl and 10mM Tris however the buffer they used to dissolved the oligos contained the wrong EDTA concentration: 25 times the amount : so 25mM rather than 1mM.

 

Anyone an idea if this would be a problem if I tried to make the oligos to anneal? 

 

 

I could add some more NaCl and Tris to dilute the EDTA , however I would have to dilute the oligos a lot than and I think they would not anneal anymore if I dilute it too a concentration that is rather low.

 

 

Now I have a 100micromolar concentration of oligos, diluting this 25 times seems a bit too much since I would end up with just 4 micro molar of the oligos.

 

Any insight?


Edited by lyok, 29 July 2014 - 10:18 AM.


#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,713 posts
398
Excellent

Posted 29 July 2014 - 12:43 PM

I suspect that the EDTA is most likely to interfere with the ligation step.  You could try a buffer exchange by dialysis, but it is probably cheaper and easier to just order new oligos.



#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,467 posts
247
Excellent

Posted 29 July 2014 - 05:03 PM

The EDTA will not inhibit the annealing, but as Bob1 says, downstream use of the oligos will likely be a problem due to the high EDTA concentration.



#4 lyok

lyok

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 206 posts
6
Neutral

Posted 30 July 2014 - 01:13 AM

Ok thanks for the information.

 

I will dilute them 10 times for the ligation, so I will end up with 2.5 mM EDTA, still more than twice what is needed (1mM) but perhaps this will not be such a big problem.

 

I will try it and if needed order new oligos.






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

©1999-2013 Protocol Online, All rights reserved.