Homemade RealTime Syb-GreenI-Mix - (Jan/27/2006 )
I have learn from sb work in company that SYBR green will inhibit PCR if you using wrong concentration, and there are also some posts in this forum stated that commercial SYBR green I reagent might contain DMSO or other solvent which will inhibit your PCR, properly you did not diluted and mix it well.
Plenty of people I know use a standard 2x PCR supermix with SYBRgreen I added at 1:10,000 conc (ie 5picolitres equivalent per 50ul PCR). The SYBRgreen is diluted in 10mM Tris just before use
We use a non-standard thermal DNA polymerase that does not have a commercially available QPCR buffer. As a result, I prepare large batches of our proprietary buffer and make 1 mL aliquots for storage at -20C.
10,000x SYBR Green I is dissolved in DMSO, but the amount of DMSO in 1x buffer is only 0.01%. (10% DMSO lowers primer Tm by 5-6°C and inhibits Taq by 50%.)
Addition of SYBR Green I may result in an increased requirement for magnesium. Titration is recommended.
thank you....now I know I am not alone :-)
ok..better mixing would be a point.....I also do the reaction with an homemade buffer and a commercial taq .
tfitzwater, maybe we can exchange our recieps :-)
I use 2mM MgCl2 as Conc in the Reaction.....25µl Reactions....
maybe I should double the volume
we use the iCycler-system
I use the Sybr Green I in a 1:20000 delution.
1:10000 results in a signal strength 10 fold higher than the commercial mix!!!!!
Roche is rumored to add a secret ingredient to their mixes which stops nucleic acids and proteins from sticking to the capillary tube walls. The 'magic ingredient' is BSA. The final concentration is supposed to be 100 ng/µL, which coats the glass capillary to prevent non-specific binding of reaction components.
Magnesium chloride concentration should be between 4 and 7 mM. It is optimized as 5.5 mM for the primers/probes designed using the Primer Express software.
does BSA also make sense when working with plastic tubes??
" trust " and "believe" - something nonscientific
for example " In God we Trust "
Persons who use this therms shurelly NOT FROM SCIENCE
Homemade sybrs are published ( see PubMED )
SYTO9 as SATURATING Dye looks better than SYBR (see PubMed)
I tried such things, too. For some time I thought it works. Until I realized that with some cDNAs (especially from brain) there was quite some inhibition. Such problems don´t appear with commercial kits. Thus, if one wants reliable results one has to pay the price (of some commercial provider).
I 've found some publication with homemade SYBR green kits, that use 30.000 fold dilutions of commercial stock from Molecular Probes (!). When I tried to do it, I get overall strong signal, also in the negative (DNA-) sample. With commercial kits i've never get any false-positive signals.
Anyway- do you always use the reference dye e.g. ROX?
Qiagen is using his HotStarTaq-Polymerase in it´s "Qiagen SYBR Green PCR Kit" (#204143).
What I´m doing is using the HotStarTaq Plus DNA Polymerase (#203603, a modified form of QIAGEN Taq DNA Polymerase) and the (included) Qiagen PCR-Buffer plus MgCl2 (also included).
What I need to add are the dNTPs (Qiagen #201900) and the dye. In his recent publication Stephen A. Bustin (Nolan, Hands & Bustin; Nature Protocols; 2006) suggests SYBR or EvaGreen. So I tried Evagreen. Because EvaGreen shows about the same excitation and emission as SYBR does you reallly don´t need to change anything in your instriment settings. Just act as if it was SYBR.
Using the following volumes for a 25µl reaction this works beautifully and I save about 50% of the costs.
add in order and mix (by pipetting up and down plus a very short vortex) before distributing the mix into the wells
Water (of course RNase and DNase free)
10x PCR-Buffer = 2.5 µl
25 mM MgCl2 = 1.5 µl (which gives a total MgCl2 concentration of 3.0 mM,(the 1xBuffer has already 1.5 mM))
dNTP-Mix (10mM of each) = 0.3 µl (which gives a concentration of 120 µM of each)
HSTaq-Plus = 0.125 µl
20x EvaGreen = 0,75 µl (this is 0.6x of manufacturers recommendation)
and don´t forget your Primer and cDNA.
1x 95°C/ 5:00;
40x (94°C/ 0:20)(Annealing°C/0:40)(72°C/0:45)
maybe anyone wants to give this a try or anyone has some recommendations.