Homemade qPCR Master Mix

Instructions

Use 0.2x (final) SYBR green in your qPCR mix. 

Notes

• You can do everything else as usual (PCR rxn setup and cycling parameters)
• SYBR green typically comes as a 10,000x stock in DMSO. You can dilute 1uL of 10,000x in 625uL of water and add 0.25uL of this dilution per 20uL qPCR rxn.
• I have done dozens of qPCR rxns with Taq, Phusion, AccuPrime and 0.2X SYBR and it has always worked.

Evidence

• Added serial dilution of SYBR green to determine the optimal amount
• Used NEB Taq and followed their procedure

Conclusions: Too much SYBR >0.33x results in no amplification. I picked 0.2x as a safe amount to add. 

Lane	1	2	3	4	5	6	7	8	9
SYBR green (x)	100bp ladder	0 (no template)	0	1.3	0.33	0.08	0.02	0.05	0.0013

PCR Details for this experiment

Component	20 μl rxn	Final Concentration
10X Standard Taq Reaction Buffer	2.0	1X
10 mM dNTPs	0.4	200 µM
Taq DNA Polymerase	0.1	1.25 units/50 µl PCR
Water	15.7
5 µM each primer (165&166)	0.8	0.4 µM (0.05–1 µM)
SYBR GreenStock: 300x dilution	varies	varies
Template DNAE coli gDNA (550ng/uL)	0.36	10ng/uL

STEP	TEMP	TIME
Initial Denaturation	95°C	30 seconds
30 Cycles	95°C 50°C 68°C	15 seconds 15 seconds 20 seconds