MicroRNA Northern Protocol
Revised 3-30-04
Reminder ­ Wear Gloves, lab coat and eye protection!!!
Gel Setup Using Protean II system
- Clean
materials with 10% SDS and rinse thoroughly with ddH20
- Set up
gel apparatus as per normal (use thick spacers) 1.5mM
- Make
up 15% Denaturing Gel
Northern Gel
|
# of Gels
|
|
40% Acrylamide
|
18.75 mL
|
5x TBE Buffer
|
5 mL
|
dH20
|
10 mL
|
Urea
|
21 g
|
10% APS
|
400 ul
|
TEMED
|
40 ul
|
- Allow gel to polymerize for 1 hour
- Assemble
gel apparatus and add the running buffer (0.5X TBE)
- Clean
out wells with running buffer ­ make sure there are NO leaks!!!
- Pre-run
the gel at 180 volts for 30 min
- Rinse
wells right before loading sample
RNA Prep and Gel Running
- You
want to load 20ug of total RNA per lane ­ add DEPC H2O up to 20uL
- Add
20uL of formamide to your RNA sample
- Heat
RNA at 65ºC for 10 min
- Chill
on ice for 1 min
- Add
some Bromophenol Blue loading dye
- Load
samples and run at 180 volts until the dye reaches the bottom of the gel
- After
running gel, stain with EtBr in 0.5X TBE for 5 min. Destain in 05X TBE.
This will allow you to visualize tRNAs and 5S RNA for normalization. Place a ruler down as a reference.
- Rinse
gel in 0.5X TBE to remove excess EtBr.
Gel Transfer with Trans-blot Semi-Dry transfer cell
- Use
the GeneScreen Plus membrane and cut to size slightly larger than the gel.
- Soak
membrane in dH20 for a few seconds
- Soak
membrane in transfer buffer (0.5X TBE) for 15 minutes
- Soak 2
pieces of whatman paper in 0.5X TBE
- Set up
transfer as such ­ From Bottom (anode) to top : whatman, membrane, gel,
whatman, cathode plate. Make
sure to roll out any bubbles
- Transfer
at 400mAmps for 1 hour. The
voltage will start out low but increase by the end of the transfer
Post Transfer
- Wash
blot in 0.5X TBE to remove any traces of the gel
- Place
wet membrane on a wet sheet of filter paper and UV Crosslink at optimal
setting
- Store
membrane at 4ºC until use
Labelling Probes
To
a screw top tube, add this
10.4ul
dH20
2ul
10x PNK Buffer
2ul
Oligo Probe
1ul
T4 PNK
5ul 32P gATP
Mix
and incubate at 37 degrees for 45 min
Add
80ul of TE
Run
through G-25 column
Count
probe
Probing Membranes
- Prehyb
membrane in Ultrahyb Oligo solution for 0.5 hours at 42º C. Add 1mL/10cm2 of
membrane.
- Add
your 32P-labeled probe to the prehyb solution and incubate 12-24 hours at
42¡C
- Pour
off hyb solution and wash membrane as follows ­ 2 washes for 30 min. in
2xSSC/0.5% SDS
- Cover
membrane in saranwrap
- Place
in phosphor-imager cassette for ~4 hours.
Stripping Membranes
Boil
membrane in stripping solution (0.1X SSC, 1% SDS) for 10-30 min
|