Protocol 13 Twostep master mix

RT-1

A - 10 |l Rxn

B - 5 |l Rxn

Stock

Component

Final

Volume (|l)

Volume (|l)

*****

DEPC-H2O

*****

2.60

1.30

10X

Rev Transcriptase Bfr

1X

1.00

0.50

2.5 mM

dNTP mix

500 |M

2.00

1.00

20 |M

Assay-specific reverse

400 nM

0.20

0.10

primer

50 U/|l

MMLV reverse

10 U/10 |il

0.20

0.10

transcriptase

6.00

3.00

RT-2

(-RT or NAC control)

10 |l Rxn

5 |l Rxn

Stock

Component

Final conc.

Volume (|l)

Volume (|l)

*****

DEPC-H2O

*****

2.80

1.40

10X

Rev transcriptase Bfr

1X

1.00

0.50

2.5 mM

dNTP mix*

500 |M

2.00

1.00

20 |M

Assay-specific reverse

400 nM

0.20

0.10

primer

6.00

3.00

1) Add 6.0 |jl (A) or 3 |jl (B) RT-1 to 3-wells/sample

2) Add 6.0 |l (A) or 3 |l (B) RT-2 to 1-well/sample

3) Add 4.0 |l (A) or 2 |il (B) total RNA/well (2.5-50 ng/|il)

4) Incubate using RT program = 50°C - 30 min; 72°C - 5 min; 20°C - hold

*-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

Was this article helpful?

0 0

Post a comment