3 replies to this topic

[wntiong]

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!

[almost a doctor]

wntiong, on Jun 28 2010, 06:55 AM, said:

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 10µl 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.

[wntiong]

almost a doctor, on Jun 28 2010, 05:31 PM, said:

wntiong, on Jun 28 2010, 06:55 AM, said:

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 10µl 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.

[sponge_nuts]

[/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!