Home brew Sybrgreen mix for QPCR

TFitzwater at Gilead.com TFitzwater at Gilead.com
Wed Apr 18 19:51:45 EST 2001


>salanti  (ali at image.dk)
>Sun 15 Apr 2001 - 13:06:03 BST


>Hi.
>Does anyone have a recipe for Sybr green mix (i.e buffers and quantities)
>for QPCR. I would also like to know if anyone has any comments on homemade

>Sybr green PCR mixes. Several companies has stressed that it is very
>important to use the ready-made Sybr green pcr mixes for QPCR, and
extremely
>suboptimal to make it yourselv!
>Thanks
>A.Salanti

I  am enclosing information from several postings at Taqman and other users
groups:

Christian        Essrich        cessrich at gladstone.ucsf.edu             via
7700taqman at listserv.it.northwestern.edu  02/22/01  reported:  "On a related
note  for custom-made SYBR buffer.  I finally succeeded to make my own well
working  SYBR  buffer by using the same 10 x Gold buffer mentioned above in
combination  with  a  recipe  from Eric Lader.   5x:  500 uL 10x PE GeneAMP
Gold  Buffer,  5  uL  10% Tween 20 (final 0.05%), 50 uL 80% glycerol (final
4%),  0.125  uL 1:100 SYBR Green I (in Type I water) (final 1:8000) and 445
uL Type I water.  This gives the same Ct's as PE's SYBR core reagents."

LightCycler Reaction Mix for the SYBR Green Assay (anonymous post at
www.nwisc.noaa.gov/protocols/memo..
A-PCRMethodsDocs/3-2000/5.11.00.22/1.html)
2 µL 10x Pwo Buffer, (4 mM final) 3.2 uL 25 mM Mg++, 9.2 uL SYBR Green
1:10,000 dilution in water plus 0.1 µg/mL, perhaps a higher dilution for
the PE 7700?, (0.2 µg total) 1 uL Primer mix, 2 uL cDNA, 0.6 uL Pwo DNA
polymerase, 18 uL Final volume

10X is 100 mM Tris-HCl pH 8.3, 500 mM KCl, 8% glycerol, 0,1% Tween 20,
1/4000 dilution of SYBR green from molecular probes. Add Mg++ to taste (3
mM). You can add FAM-ROX oligo from Synthegen to 600 nMole in the 10X if
you want to include the reference. (Eric Lader elader at ambion.com 11.23.00)

Applied Biosystems told us that their SYBR Green master mix contains an
enhancer. "Ralf Steinborn" (ralf_steinborn at hotmail.com via
owner-7700taqman at listserv.it.northwestern.edu 11.23.00 )
Perhaps 100 ng/uL BSA?

Concerning homemade SyBr Green reagents, we tested a couple of different
protocols and found that a published one works as well as the PE mix.  You
can find it in BioTechniques, Vo. 24(6), p. 954.  We substitute HotStarTaq
from Qiagen instead of Taq plus antibody. We use it with the 2-step 95-60
protocol advised by PE instead of the three step protocol in the paper. We
also use 300nM of each primer instead of 5 micromolar.  We also have had
good success using the PrimerSelect program (part of LaserGene) to find
primers that fit the length and Tm recommendations of PE. (David Fruman
dfruman at uci.edu Sent by: owner-7700taqman at listserv.it.northwestern.edu
12/13/00 04:17 PM)

Tim Fitzwater
Gilead Sciences

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