LABELLING, HYBRIDIZATION AND DETECTION OF NON-RADIOACTIVE
There are several different ways of adding label to a DNA probe, including
the three most common: end-labeling, Nick translation and Random prime
labeling. I will be using Random Prime labeling to add 32P
labelled d-CTP to DNA strands. In lab we will use Photobiotin to label
DNA. Photobiotin is activated by light and binds covalently to DNA.
Photoactive Biotin Labeling
- The photoactive biotin (PAB) is light sensitive and should be used
under subdued light. A 100 ng/Ál solution will be available in the
lab. Mix all of your gene cleaned DNA fragment with PAB in a 1:3 (w/w)
ratio. Place the mixture in a 1.5 ml microcentrifuge tube and place
in an ice bucket; keep the lid of the tube open.
- Place a 275-watt sunlamp 10 cm above the tubes. Irradiate for 15
minutes. It is essential to keep the ice bucket filled with ice to
avoid ignition of the bucket.
- Add a volume of 0.1 M Tris-HCl, pH 9.00 that is equal to the volume
of your DNA plus photobioitin and then add 100 Ál of nH2O.
- Extract the unincorporated PAB with an equal volume of water-saturated
butanol. CAUTION: The aqueous phase - the one you want to save -is
the LOWER layer. Repeat the extraction.
- Add 1/10 volume of 3 M sodium acetate (pH 5.2) and 3 volumes of
cold 100% ETOH. Place at -20oC until ready for use.
- Pellet the DNA by centrifugation at 14,000 RPM for 15 minutes at
4oC. Wash the pellet once with 70% ETOH.
- Dry the pellet and resuspend in 500 Ál nH2O.
Prehybridization and Hybridization
Pre-Hybridization /Hybridization Solution
Amount/ 120 ml
5X Denhardt's solution
Boil salmon sperm DNA for 10 minutes and then place on ice. Add 600 Ál
of the boiled DNA to 60 ml of the above solution. Place the solution in
a plastic container. Add the northern blot, southern blot and the filters
for your cDNA library screen one at a time. Be sure that each filter and
membrane is totally submerged before adding the next. Seal the plastic
container and place it in the shaking incubator at 65oC for
2 hours - overnight.
Add the hybridization solution to a 60 ml conical tube, add your biotinylated
DNA to this and boil for 10 minutes. Remove the pre-hybridization solution
and filters from the plastic container. Place the filters on the lid of
the container. Place the boiled solution in the container and add the
membranes and filters one-by-one, making sure each is completely covered
with solution. Seal the container and place in the 65oC incubator
Washes and Detection
- Wash 2X in 2X SSC, 0.5% SDS for 10 minutes at 65oC.
- Wash once in 0.2 X SSC, 1% SDS for 10 minutes at 65oC.
- Wash the blots and filters in blocking solution for 30 minutes shaking
gently. This step prevents most of the non-specific binding of the
strepavidin alkaline phosphatase (SAP) conjugate to the nitrocellulose
- Remove the liquid and add SAP conjugate (10 ml Buffer A containing
25 Ál of conjugate). Incubate for 25 minutes, shaking gently.
- Wash 3X for 10 minutes each with 50 ml of Buffer A.
- Wash for 5 minutes with Buffer C.
- Incubate in dye solution under reduced light for 30 minutes to 3
- When blue-black reaction product is seen, stop the reaction by washing
the blot with 1.0 mM EDTA.
- Store away from strong light after drying.
1.0 M NaCl
1 M Tris-HC L, pH 7.5
2.0 mM Mg Cl2
0.05% Triton -X 100
Blocking Solution: Buffer A plus 3% bovine serum albumin
0.1M Tris-HC L, pH 7.5
10 mM Mg Cl2
64 Ál of NBT (nitro-blue tetrazolium) (50 mg/ml)
32 Ál BCIP (5-bromo-4-chloro-3-indolyl phosphate)
10 ml Buffer C