DIG Random primed DNA labelling and detection of hybrids using NBT/BCIP

DIG RANDOM PRIMED DNA LABELLING AND DETECTION OF HYBRIDS USING NBT/BCIP

Last Update: December 2006

PREPARE SOLUTIONS

1. Buffer 1: Maleic acid buffer (100 mL):

Mix 1.38 g of maleic acid (0.1M), 1.5 mL of 10 M NaCl (0.15M), and dH₂O to 100 mL. pH to 7.5 with solid NaOH

2. Buffer 2: blocking solution (100 mL):

Mix 10 mL Blocking solution with 90 mL maleic acid buffer

3. Buffer 3: detection buffer (100 mL):

Mix 10 mL of 1M Tris-HCl, pH 9.5 (0.1M), 1 mL of 10 M NaCl (0.1M), and 1 g of MgCl₂ (50mM). pH to 9.5

4. Antibody solution (anti-DIG-AP):

Dilute 1:2,000 - 1:5,000 in buffer 2

5. 100X NBT (5 mL):

Mix 150 mg of NBT (chloride), 3.5 mL of DMF, and 1.5 mL of dH₂O. Store in the dark at 4^oC

6. 100X BCIP (5 mL):

Mix 75 mg of BCIP with 5 mL of DMF. Store in the dark at 4^oC

7. NBT/BCIP

40 mL in 2 mL of buffer 3

8. Standard hybridization buffer (100 mL):

Mix 25 mL of SSC (5X), 0.1 g of Sarcosyl (0.1%), 0.2 mL of 10% SDS (0.02%), and dH₂O to 90 mL. Before use: add 10 mL Blocking solution

PROCEDURE - Step 1 - DIG DNA Labeling

NOTE: it is recomended to test the titer of the probe before using it. Alternatively, if the PCR worked, all the reaction can be used and test directly on the hybridization

1. Add 1 mg of DNA in 16 mL of dH₂O to an eppendorf tube (prepared as described; 108. DNA Fragment Isolation from LM agarose, 194. PCR DNA product purification (Qiagen); 195. DNA extraction from agarose (Qiagen))

2. Denature the DNA by heating in boiling water for 10 mins and quickly chiling on ice (ice/ethanol)

3. Add 4 mL of DIG-High Prime, mix and centrifuge

4. Incubate for 1-20 hours at 37^oC

5. Stop reaction by adding 2 mL of 0.2 M EDTA (pH 8.0) and/or heating at 65^oC for 10 mins

NOTE: there is no need to purify the product from the unincorporated DIG-dUTP

PROCEDURE - Step 2 - Hybridization for Southern blots

1. Prewarm (to desired temperature: 45-65^oC) appropiate amount of Standard hybridization solution (20 mL / 100 cm²) and incubate filter for 30 mins with gentle agitation

2. Denature Dig-labeled probe by boiling for 5 mins and rapidly cooling on ice water

3. Add to prewarm hybridization solution (2.5 mL /100 cm²) and mix well (AVOID bubbles)

4. Pour off prehybridization solution and add probe-hybridization solution mix to membrane

5. Incubate with gentle agitation for at least 6 hours

PROCEDURE - Step 3 - Washes

1. 2X for 5 mins in 2X SSC, 0.1% SDS at RT

2. 2X for 15 mins in 0.1X SSC, 0.1% SDS at 65^oC

Probe reuse: DIG-probes can be stored at -20^oC and reused many times as long as they are first denatured at >68^oC

PROCEDURE - Step 4 - Immunological detection (assuming a 10 x 10 cm blot)

1. After washes, rinse briefly (1-5 mins) in maleic acid buffer

2. Incubate for 30 mins in 100 mL blocking solution

3. Dilute anti-DIG-AP conjugate 1:5,000 in blocking solution

4. Incubate membrane for 30 mins in 20 mL antibody solution

5. Wash 2X for 15 mins with 100 mL maleic acid buffer

6. Equilibrate for 2-5 mins in 20 mL detection buffer

7. Incubate in 10 mL freshly prepared color solution, sealed in a plastic bag or box in the dark (DO NOT SHAKE)

8. Color develops within a few minutes and the reaction is complete in <16 hours. Expose membrane to light only to monitor color development

9. Stop reaction by washing for 5 mins with water or TE

PROCEDURE - Step 5 - Stripping and reprobing of DNA blots

1. Heat a large beaker with DMF to 50-60^oC in a water bath

2. Incubate the membrane in the DMF until the blue color has been removed (changing the DMF may speed the process)

3. Rinse membrane throughly with dH₂O

4. Wash 2X for 20 mins at 37^oC in 0.2M NaOH and 0.1% SDS to remove DIG-lalbeled probe

5. Rinse throughly in 2X SSC

6. Rehybridize and hybridize with second probe