|
Reagents:
20X
SSC
(3M NaCl, 0.3M NaCitrate, pH 7.0):
| |
1000ml |
2000ml |
| NaCl |
175g |
350g |
| Na3citrate.2H2O |
88g |
176g |
-
adjust pH to 7.0 with 1M HCl
100X
Denhardt:
| |
100ml |
500ml |
| Ficoll-400 |
2g |
10g |
| Polyvinyl-pyrrolidone |
2g |
10g |
| Bovine
Serum Albumin |
2g |
10g |
| ddH2O |
to 100ml |
to 500ml |
-
filter sterilize
-
store at –20 °C in 25ml aliquots
Hyb
Solution (5X
SSC, 5X Denhardt, 50% Formamid, 1% SDS)
| |
5ml |
10ml |
15ml |
20ml |
| 20X
SSC |
1.25ml |
2.5ml |
3.75ml |
5.0ml |
| 100X
Denhardt |
0.25ml |
0.5ml |
0.75ml |
1.0ml |
| di-Formamid |
2.50ml |
5.0ml |
7.50ml |
10.0ml |
| 10%
SDS |
0.50ml |
1.0ml |
1.50ml |
2.0ml |
| ssDNA
(11mg/ml) |
50ml |
100ml |
150ml |
200ml |
| ddH2O |
0.45ml |
0.9ml |
1.35ml |
1.8ml |
Protocol:
- Rinse gel with ddH20, 3X.
- Soak in 20X SSC for 45 minutes.
- Take photograph of gel.
- Soak nylon membrane -cut to size- in ddH2O
for 5 minutes.
- Put sponge in container, fill with 20X SSC
halfway up sponge.
- Put 3 20X SSC-soaked GB002-sheets on top of
sponge.
- Place gel on top, remove bubbles.
- Cover with nylon membrane, remove bubbles.
- Successively add 1 GB002, 4 GB003 and 4cm GB004.
- Cover with glass plate and 0.4kg weight.
- Transfer overnight.
- Take structure apart, mark wells on membrane.
- Visualize and mark rRNA bands and RNA MW ladder
on membrane.
- Rinse membrane in 2X SSC.
- Dry on GB003 and wrap in plastic foil
- UV cross-link (“Autolink” on Stratalinker).
- Take photograph of flattened gel to assess
transfer efficiency.
- Pre-hybridize membrane for 4-20 hours in 5-10 ml
Formamid (Pre-) Hybridization solution (FPH) at 42 °C.
- Boil 100µl probe for 10 minutes (use 1 x 106
cpm/ml FPH).
- Cool on ice, spin.
- Add to hybridization bag.
- Incubate 20 hours at 42 °C.
- Rinse in 2X SSC/0.1% SDS at RT, 3X.
- Wash in 0.2X SSC/0.1% SDS for 15 minutes at 42 °C,
2X.
- Wash in 0.1X SSC/0.1% SDS for 15 minutes at 65 °C,
2X.
- Rinse in 2X SSC.
- Wrap in plastic foil.
- Expose to phosphoscreen.
Notes:
-
After overnight exposure 5pg RNA can be detected
with a probe labeled to a specific activity of 109 dpm/mg.
-
Probes labeled to ≥5
x 108 dpm/µg should detect transcripts that
represent 0.01% of mRNA population with a blot of 10 µg
total RNA or 0.0002% of mRNA population with a blot of
3 µg poly(A)+ RNA.
-
For stripping poor boiling 0.05% SDS on membrane,
incubate for 10 minutes, repeat up to 3X. Rinse with 2X
SSC.
For specifics on appropriate handling and waste procedures please see the
online chemical SOPs or our waste and spill notebook located in
room 352.
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