-
Linearize 10 µg
of plasmid using the appropriate restriction enzyme - do this for both
antisense and sense probes.
- Stop the
reaction by heating the DNA at 65°C for 10 minutes.
- Purify plasmid
DNA by adding equal volume of 50% chloroform/50% phenol
(spin down for 5 minutes at room temperature and remove
top layer).
- Ethanol
precipitate (for a 20 µl
add 2.2 µl
of NaOAcetate and 20 µl
EtOH,
chill at -20°C for at least 30 minutes,
spin for 15 minutes (at maximum speed) and
re-suspend pellet in 20µl
DEPC'ed H2O.
- Using Megascript
Kit to make RNA.
- Thaw 10X
transcription buffer, ribonucleotide solutions and RNAase-free water,
vortex, and microfuge to remove solution from the
caps of the tubes -- WEAR GLOVES!
- Add the
following amounts of the indicated reagents in the order below
to a 1.5 µl
RNase
free microfuge tube at room temperature
2 µl
10X transcription buffer
2 µl
Ribonucleotide mix (10mM ATP, CTP, GTP, UTP & digoxigenin-UTP {2:1})
_µl
Linearized plasmid DNA (1µg)
(e.g. 2 µl
of a
0.5 µg/ml
template).
2 µl
Enzyme mix
_ µl
RNAase free water (final volume of reaction will be 20 µl)
Mix and spin
down
8. Incubate
in a cabinet 37°C incubator for between 2 to 6 hours
(if you do not have time to finish, freeze the reaction and
resume the next day
- do not let the reaction go overnight!).
At this point you can run (on a 1% MOPS RNA gel) 1µl
of the reaction to see if you have probe.
9. Add 1 µl
RNAase-free, DNAase I (2U/µl),
mix, spin and incubate at 37°C for 15 minutes.
10. Add 1/3
volume of 10 M LiCl in DEPC'ed H2O mix thoroughly
- chill
reaction for at least 30 minutes at -20°C
9. Centrifuge
at 4°C for 25 minutes at maximum speed to pellet the RNA,
carefully remove the supernatant and wash the pellet
with 70% ethanol.
Centrifuge at 4°C for 10 minutes at max speed to
remove nucleotides.
10. Remove
as much supernatant as possible being
careful not to dry the pellet too much.
Resuspend in 20-100 µl
DEPC-water and store at -80°C. |