Northern Blot With DIG
updated 05/05/05 - Benjamin Costantino
(For the PDF version, click here.)
Labeling
1. DNA template (10ng - 30µg) to 15µl and boil 100° C for 10 min.
2. Snap cool on ice for 5 min.
3. Transfer: (DIG-DNA Labeling Kit: Roche #11 175 033 910)
a. 2µl 10x Hexanucleic mix (#5)
b. 2µl dNTP Label mix (#6)
c. 1µl Klenow (#7)
4. Total of 20µl, mix.
5. Place tube in 37°C up to overnight.
6. Kill Klenow by heating 65°C for 10 min.
7. Add up to 200µl; spin purify with G50 Sephadex 2min. @ 1000rpm; collect in screw cap tube.
Hybridization
1. Preheat EasyHyb ~ 40mls at 50°C.
2. Gently agitate blot with ~30mls preheated EasyHyb for 30 min. (minimum)
3. Denature DIG-DNA probe and salmon sperm DNA (final conc. 100µg/ml) - boil 10min. and then Snap cool on ice for 5min.
4. Add DIG-DNA probe and salmon sperm DNA to 5mls of preheated EasyHyb.
5. Dump prehybridization and transfer blot to seal-a-meal bag.
6. Add 5ml of EasyHyb/Probe to bag.
7. Incubate overnight at 50°C.
Wash
1. Discard EasyHyb and transfer blot to tray containing 30ml of 2 x SSC/0.1% SDS (Low Stringency Buffer) - 2 x 5min. rotating.
2. Discard Low Stringency Buffer; Add 30ml of .1 x SSC/0.1% SDS (High Stringency Buffer) (preheated at 50°C) - 2 x 15min. rotating @ 50° C.
3. Discard High Stringency Buffer; Add 50ml of 0.1M Maleic Acid / 0.15M NaCl / 0.3% Tween 20 - pH7.5 (Wash Buffer) - 2min. rotating.
4. Discard Wash Buffer; Add 40ml of 1:10 Block (Roche #11096176001) : 0.1M Maleic Acid / 0.15M NaCl pH7.5 (Blocking Solution) - 1 hr. rotating.
5. Discard Blocking Solution; Add 40ml of Antibody (αDIG-POD Roche #11207733910) @ 1:5000 : Blocking Solution - 30min rotating.
6. Discard Antibody/Block Solution; Wash membrane 2 X 15min with 30ml of Washing Buffer.
7. Rinse in 1x PBS until ready to detect.
Detection
1. Mix 1:40 of luminol substrate. (Amersham #RPN2132V1/V2)
2. Pour onto membrane and cover in dark for 2min.
3. Transfer membrane to fresh saran wrap and expose film O/N.
4. Process film through the developing machine.
Notes
A. Make a Sephadex Column:
- Mix 2g G50 sephadex in 50ml in 1x TE.
- Autoclave to fully hydrate.
- Fill 1ml syringe stuffed with glass wool or cotton plug.
- Spin 1000rpm for 2min to pack sephadex.
B. Maleic Acid pH:
- pH to 7.5 using solid NaOH.
