Protocol 14 Twostep PCR master mix for probebased assays

PCR-1 Stock

Component

Final conc.

50 |l Rxn Volume (|l)

25 |l Rxn Volume (|l)

*****

PCR-H2O

*****

23.20

11.60

10X

PCR Bfr

1X

5.00

2.50

15 |M

SuperROX™ (Biosearch)

90 nM

0.30

0.15

50 mM

MgCl2

5 mM

5.00

2.50

2.5 mM

dNTP mix*

200 |M

4.00

2.00

20 |M

Forward primer (+)

400 nM

1.00

0.50

20 |M

Reverse primer (-)

400 nM

1.00

0.50

20 |M

Probe

100 nM

0.25

0.13

5 U/|l

Taq (regular or hot-start)

1.25 U/50 |l

0.25

0.13

40.00

20.00

1) Add 40 |jl (A) or 20 |jl (B) PCR-1 to each of 4 wells cDNA/assay

2) Use the following cycle program:

95°C - 1-15 min; 40 cycles of 95°C - 12-15 sec, 60°C - 30-60 sec

*-2.5 mM dNTP mix - equal volumes of the 4 dNTPs at 10 mM

Standard RT master mix from the Quantitative Genomics Core Laboratory; UTHSC-Houston, Houston, TX 77030, USA

An introduction to real-time PCR 37

Protocol 1.5: Two-step PCR master mix - primer or SYBR® Green I based assay

PCR-1

50 |l Rxn

25 |l Rxn

Stock

Component

Final conc.

Volume (|l)

Volume (|l)

*****

PCR-H2O

*****

12.70

6.35

10X

PCR Bfr

1X

5.00

2.50

15 |M

SuperROX™ (Biosearch)

90 nM

0.30

0.15

40%

Glycerol

8%

10.00

5.00

10%

Tween 20

0.10%

0.50

0.25

50 mM

MgCl2

5 mM

5.00

2.50

2.5 mM

dNTP mix*

200 |M

4.00

2.00

20 |M

Forward primer (+)

400 nM

1.00

0.50

20 |M

Reverse primer (-)

400 nM

1.00

0.50

20 |M

Probe

100 nM

0.25

0.13

5 U/|l

Taq (regular or hot-start)

1.25 U/50 |l

0.25

0.13

40.00

20.00

1. Add 40 |jl (A) or 20 |jl (B) PCR-1 to each of 4 wells cDNA/assay

2. Use the following cycle program:

95°C - 1-15 min; 40 cycles of 95°C - 12-15 sec, 60°C - 2 sec, 72°C - 30-60 sec

*-2.5 mM dNTP mix - equal volumes of th 4 dNTPs at 10 mM

Standard RT master mix from the Quantitative Genomics Core Laboratory; UTHSC-Houston, Houston, TX 77030, USA

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