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
- - - - -

PCR troubleshoot

pcr

  • Please log in to reply
4 replies to this topic

#1 kidakarn

kidakarn

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 31 August 2014 - 05:37 PM

Hello, Im a graduate student. Now, I working on part of PCR lab. The problem is I use the condition that my adviser was use and it give a good result with a thick band of PCR product but last two week I perform the experiment with the same protocol as previous and I got the faint band. So help me please.



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,676 posts
396
Excellent

Posted 31 August 2014 - 08:45 PM

Most likely one of the components has gone off, probably the dNTPs. Make fresh aliquots of the dNTPs, the primers and your template DNA and test them.



#3 kidakarn

kidakarn

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 05 September 2014 - 05:15 PM

Thank you a lot,

 

However, now I use new master mix and also order a new set of primer.

When I use 25 ul/reaction and load 5 ul into gel it give a good band but when I make 100 ul/reaction band is faint than 25 ul/reaction.

help me please.



#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,676 posts
396
Excellent

Posted 06 September 2014 - 11:03 AM

So are the concentrations of reagents different between the two volumes? How much DNA do you add to each? Are the cycling conditions the same? Are you mixing properly?



#5 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,765 posts
131
Excellent

Posted 08 September 2014 - 03:46 AM

keep in mind that the thermal characteristics of 100ul are different from those of 25ul. you may want to try making 4 X 25ul reactions and combining them after cycling.


talent does what it can
genius does what it must
i do what i get paid to do





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

©1999-2013 Protocol Online, All rights reserved.