Real-Time PCR buffers

Cynthia syntheticamatrix at yahoo.com
Tue Oct 9 18:39:50 EST 2001


Genger <> wrote in message news:<20010926111716.31845.qmail at ww02.jatek.com>...
> Hi all.
> I am trying to make my own PCR buffers for Real-time detection on the 5700
> SDS GenAmp system. I can't afford to by original reagents from AP Biosystems
> due to the incredible high price. I have some idea but havn't any idea about
> passive references in TaqMan A and SYBR Green buffers. Some one wrote me
> about ROX but I got a PCR ingibition effect by using ones. I would greatly
> appreciate for any advise from you.
> HELP! Dont let me go bankrupt!!!
> 
> 
> <http://www.biowww.net/forum/read.php?f=1&i=3926&t=3926>
Hello,

I have heard of one company that services people wanting to save money
on mixing their own PCR mix: EpiCentre. I believe it was
epicentre.com, but maybe a Google search is in order if that doesn't
work.

The passive reference for ABI is to normalize the signal.  Some use
ROX dye straight, but this can fall out of solution and cause
"ROX-drop." A mixture of 0.01% Gelatin and 0.01% Tween20, may cheaply
keep the ROX from dropping, but, usually the ROX is in the form of a
tiny non-specific oligo with a 3'FAM label and a 5'ROX label.  The FAM
works as an "antenna" for the ROX, so it is excited even though 490nm
laser is on the 5700.

Sybr-Green can be purchased at Molecular Probes.  I think it's cheaper
there: www.probes.com.

When you say Buffers, do you mean the recipe for TE Buffer? If so, I
have that handy.

Do you need a cheap source for probes and primers?

I hope this helps,
Cynthia




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