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
- - - - -

how to save sybr green reagents?


  • Please log in to reply
3 replies to this topic

#1 wntiong

wntiong

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 58 posts
0
Neutral

Posted 27 June 2010 - 09:55 PM

hi,

i'm having short of funding and resource problems. i only have 500 sybr green reactions left and i need to screen for 4 genes of interest and 1 housekeeping gene for 30 samples. Still waiting for my primers to arrive, but previous batch of primers i tested doesn't compatible each other, that means i can't use delta delta ct method. Problems now are i don't have much reactions left, and if i run duplicate and use standard curve method everytime, for about 15 samples i'll need 700 reactions. This is not included the failure of experiment if there is NTC amplification or for primer validation test. Tell me what should i do. One way to save the reaction i thought will be not to run all my experiments in duplicate.
Need your all advise. Thanks!

#2 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 28 June 2010 - 01:31 AM

hi,

i'm having short of funding and resource problems. i only have 500 sybr green reactions left and i need to screen for 4 genes of interest and 1 housekeeping gene for 30 samples. Still waiting for my primers to arrive, but previous batch of primers i tested doesn't compatible each other, that means i can't use delta delta ct method. Problems now are i don't have much reactions left, and if i run duplicate and use standard curve method everytime, for about 15 samples i'll need 700 reactions. This is not included the failure of experiment if there is NTC amplification or for primer validation test. Tell me what should i do. One way to save the reaction i thought will be not to run all my experiments in duplicate.
Need your all advise. Thanks!


Which reaction volume are you using? and which qRT-PCR instrument? A simple way I can think of is halving your reaction volume, I've used as little as 10Ál in the past. Of course this depends on your instrument you are using, and you'll have to be very very careful and precise with your pippetting to avoid introducing extra errors.

#3 wntiong

wntiong

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 58 posts
0
Neutral

Posted 28 June 2010 - 09:36 PM

hi,

i'm having short of funding and resource problems. i only have 500 sybr green reactions left and i need to screen for 4 genes of interest and 1 housekeeping gene for 30 samples. Still waiting for my primers to arrive, but previous batch of primers i tested doesn't compatible each other, that means i can't use delta delta ct method. Problems now are i don't have much reactions left, and if i run duplicate and use standard curve method everytime, for about 15 samples i'll need 700 reactions. This is not included the failure of experiment if there is NTC amplification or for primer validation test. Tell me what should i do. One way to save the reaction i thought will be not to run all my experiments in duplicate.
Need your all advise. Thanks!


Which reaction volume are you using? and which qRT-PCR instrument? A simple way I can think of is halving your reaction volume, I've used as little as 10Ál in the past. Of course this depends on your instrument you are using, and you'll have to be very very careful and precise with your pippetting to avoid introducing extra errors.


@almostadoctor,

Thanks! I'm using rotorgene 6000 machine and rotorgene qpcr sybr green mastermix, the minimal volume the kit required is 25ul.

#4 sponge_nuts

sponge_nuts

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 04 August 2010 - 07:54 AM

[/quote]

@almostadoctor,

Thanks! I'm using rotorgene 6000 machine and rotorgene qpcr sybr green mastermix, the minimal volume the kit required is 25ul.
[/quote]

Hi there,
I used to used a Rotorgene 3000 and managed to scale down the volume reaction to 15 ul, even if the recomended volume was 25 ul. You should have a go!




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

©1999-2013 Protocol Online, All rights reserved.