First Time , Real-Time PCR HELP!!!

[c1bl]

Hey,

I'm just starting out with this qPCR stuff. I'm looking for for protocols which don't use kits. I can't seem to find any online. I know you use SYBR green as the dye however all the protocols i see use SYBR green kits from Qiagen or Applied biosytems ect. I'm wondering where to get eh SYBR green dye and how much to add to my reactions. Also will I have to make the PCR buffer too? If so do you recommend just getting the SYBR green Kit from Qiagen.

My Supervisor doesn't like kits because they cost a little bit more but do you think it's worth it in the end. Less work and more reliable results.

Thanks,
Chris

[̑̑̑]

i have never heard of do-it-yourself qpcr, i've always used sybr green kit from abi and it worked fine for me... but who knows maybe there is sumething out there... check the board, there seems to be a fair amount of people into the technique, apart from that, google seems to be of great help whenever i need sumething... hope it helps man

[migandi]

Hello

[danfive]

Originally Posted by c1bl

Hey,

I'm just starting out with this qPCR stuff. I'm looking for for protocols which don't use kits. I can't seem to find any online. I know you use SYBR green as the dye however all the protocols i see use SYBR green kits from Qiagen or Applied biosytems ect. I'm wondering where to get eh SYBR green dye and how much to add to my reactions. Also will I have to make the PCR buffer too? If so do you recommend just getting the SYBR green Kit from Qiagen.

My Supervisor doesn't like kits because they cost a little bit more but do you think it's worth it in the end. Less work and more reliable results.

Thanks,
Chris

First your supervisor is messing with you SYBR green is almost always sold as a kit with Taq, dNTPs and PCR buffer in it (see sample recipe).
Secondly if somebody is suggesting you use a conventional PCR recipe (no Supermix/Readymix) and just add SYBR green to make it a qPCR recipe---then make sure to buy this one http://probes.invitrogen.com/media/publications/146.pdf My point is that it is SYBR green I dye, not SYBR green II.

Here is how I use BioRad SYBR Green Supermix

Sample PCR rxn recipe:
SYBR Green Supemix 7.5ul
Primer 1 0.2
Primer 2 0.2
H2O 5.6
DNA 1.5
-----
15ul
primers final conc 0.15uM

Hope this helps,

[Jeanyo1ogy]

Your supervisor must place very little value on your time, not to mention the samples you will burn through in trying to optimize this approach. There are times where it just makes sense to use the proven tools out there. If you plan to publish, using some a homebrew mix is just going to be one more variable to be concerned about. You're not supposed to build your own thermal cycler too, I hope!

Just my $0.02 worth.

[Edi]

Hi Chris,

Hope this helps:
SYBR premix ex Taq (+ ROX dye) 10uL
PCR fwd primer 0.4 uL
PCR rev primer 0.4 uL
DNA 2 uL
dH20 6.8 uL
----------------------------------------
20 uL
final primer con = 10 uM

I'm afraid those are from Takara-Bio kit that I use on ABI Prism 7300.

Good luck.