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Tutrorial

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Please direct all comments, questions, and suggestions to biobzc@hofstra.edu

LABELLING, HYBRIDIZATION AND DETECTION OF NON-RADIOACTIVE PROBES

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

    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. 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.
    6. Pellet the DNA by centrifugation at 14,000 RPM for 15 minutes at 4oC. Wash the pellet once with 70% ETOH.
    7. Dry the pellet and resuspend in 500 Ál nH2O.

Prehybridization and Hybridization

Pre-Hybridization /Hybridization Solution

Ingredient

Stock

Amount/ 120 ml

5X Denhardt's solution

50 X

 

5X SSC

20 X

 

0.5% SDS

10 %

 

Water

   

TOTAL

 

120 ml

Prehybridization

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.

Hybridization

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 overnight.

Washes and Detection

    1. Wash 2X in 2X SSC, 0.5% SDS for 10 minutes at 65oC.
    2. Wash once in 0.2 X SSC, 1% SDS for 10 minutes at 65oC.
    3. 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 and nylon.
    4. Remove the liquid and add SAP conjugate (10 ml Buffer A containing 25 Ál of conjugate). Incubate for 25 minutes, shaking gently.
    5. Wash 3X for 10 minutes each with 50 ml of Buffer A.
    6. Wash for 5 minutes with Buffer C.
    7. Incubate in dye solution under reduced light for 30 minutes to 3 hours.
    8. When blue-black reaction product is seen, stop the reaction by washing the blot with 1.0 mM EDTA.
    9. Store away from strong light after drying.

Solutions

Buffer A

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

Buffer C

0.1M NaCl
0.1M Tris-HC L, pH 7.5
10 mM Mg Cl2

Dye Solution

64 Ál of NBT (nitro-blue tetrazolium) (50 mg/ml)
32 Ál BCIP (5-bromo-4-chloro-3-indolyl phosphate)
10 ml Buffer C