Generate Probes [ download protocol ]

To generate labeled RNA probes, we first incorporate a T7 promoter into the probe template using PCR followed by in vitro transcription using biotin labeled UTP.

A. T7 Enrichment PCR

Dilute templates ~500-fold to <1nM
Set up PCR reactions on ice for sense and antisense probes.


IVT Mix

1 Reaction

Diluted Enriched DNA

1μl

T7 Primer Mix (25uM)

0.5 μl

H2O

11.5 μl

Phusion HF 2x Master Mix

12.5 μl

Total

25 μl

PCR:  98C 30 seconds, 3x [98C 10s, 62C 20s, 72C 20s], 11x [98C 10s, 68C 20s, 72C 20s] 72C 60 s, 4C hold.
Clean-up with 20uL SILANE beads.  Elute in 20uL H2O.

B. In Vitro Transcription

Set up IVT reaction:


IVT Mix

1Reaction

DNA template (~250ng) + H2O

11.3μl

10 × T7 Transcription Buffer

2μl

20mM ATP

1μl

20mM CTP

1μl

20mM GTP

1μl

20mM UTP

  0.75 μl

10mM 16-Biotin UTP

0.5μl

T7 RNA Polymerase

2μl

100mM DTT

0.25μl

RNase Inhibitor

0.2 μl

Total

20μl

Mix well by pipetting.
Incubate at 37°C overnight (4+ hours).

C. DNase Treatment