Does anyone have a good recipe for homemade Sybr Green Master mix? I have tried a couple, and to my understanding the brand/variant of the DNA polymerase is a major factor. Any experiences as to which polymerase works the best?
I have to agree with Thomas here. Many real time PCR protocols are moving away from SYBR Green because it inhibits PCR at higher concentrations. People are turning to what are called "saturating dyes" because you can use these dyes at high enough concentrations to saturate the double strand DNA with dye molecules. This is especially great if you want to do High Resolution Melting. Examples of these dyes are EvaGreen (Biotium, Biorad) and BRYT Green (Promega). Using a saturating dye may make your master mix less dependent on the type of DNA Polymerase.
I believe that the issue is that Sybr green inhibits Taq, and to a lesser extent, may alter the available amount of Mg++. This is apparently somewhat less of an issue with the newer generations of DNA indicator dyes (e.g. EvaGreen); thus they can use more dye (which means more signal).
Here's a manufacturer's note that may provide a decent starting place for optimization: http://www.spartanbio.com/wp-content/themes/spartan/assets/application_notes/9.pdf
Mix Conc. ul
h2o 15.15
Buffer 10 X 2.5
MgCl 50 mM 1.5
dNTPs 10 mM 0.5
Primers 5 uM 2
BSA 10 mg/ml 0.25
Taq plat 5 U/ul 0.2
Sybr (10x) 10 X 0.4
Rox (5x) 50 X 0.5
Total: 23
cDNA 2
I have tried the platinum Taq from Invitrogen and another similar from Fermentas and both worked very well with the above recipe using StepOne machine from Applied. However, I had to increase SYBR and decrease ROX concentrations for Stratagene Mx3005P and iCycler from BioRad machines. Hope It helps.
Http://www.kapabiosystems.com/products/name/kapa-sybr-fast-qpcr-kits
say good bye to all optimization.
I have to agree with Thomas here. Many real time PCR protocols are moving away from SYBR Green because it inhibits PCR at higher concentrations. People are turning to what are called "saturating dyes" because you can use these dyes at high enough concentrations to saturate the double strand DNA with dye molecules. This is especially great if you want to do High Resolution Melting. Examples of these dyes are EvaGreen (Biotium, Biorad) and BRYT Green (Promega). Using a saturating dye may make your master mix less dependent on the type of DNA Polymerase.
I see that Biotium sells EvaGreen dye as a separate product from their qPCR kits, http://www.biotium.com/product/product_types/search/price_and_info.asp?item=31000
but I can't seem to find if Promega sells a BRYT Green solution.
You're right, David, The Promega dye is part of the GoTaq qPCR Master Mix and is not available separately.
@Karen Reece
Can you say whether BRYT green is different than the dye in the Quantifluor kit?
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