Molecular Analysis of Mitochondrial DNA Point Mutations by Polymerase Chain Reaction - Clinical Applications of PCR - page 140

Table 4
Setup of Dot Blots

Normal Mutant

3243nl A3243G
T3271C
G3460A
8344nl A8344G
T8356C
G8363A

8993nl T8993G

T8993C

11778nl G11778A

14459nl G14459A

14484nl T14484C

2. Prewarm Church pre-hybridization and hybridization buffer and sheared salmon

sperm DNA at 65°C until completely dissolved.

3. Place the filters in 15-mL screw top conical tubes (label tubes same as filters).

4. Add 3 mL Church hybridization buffer and sheared salmon sperm DNA stock

solution (10 mg/mL) to a final concentration of 100 µ g/mL.

5. Incubate filters at 65°C for 30 min.

3.6. End Labeling of ASO Probes With T4 Polynucleotide Kinase
1.

Each labeling reaction is 3.75 µ L and contains 2.75 µ L of master mix and 1 µ L of

a 2 M ASO probe. Depending on the number of probes to be labeled, adjust the

total volume of master mix accordingly. For example, if there are 17 probes to be
labeled, multiply the volume of each reagent by 17.

µ

2. The master mix for one probe labeling is prepared as follows:

a. 0.375 µ L 10X T4 Kinase Buffer.

b. 0.375 L T4 Polynucleotide Kinase enzyme (10,000 U/mL from New England

Biolabs).

µ

c. 0.75 µ L γ -P 32 ATP (4500 Ci/mmol, 10 µ Ci/ µ L, total amount is 2.2 p moles) (ICN).

d. 1.25 µ L sterile H 2 O.

3. Label the 0.5-mL microcentrifuge tubes according to the name of the blot as

listed under Subheading 3.4.

4. Aliquot 2.75 µ L of master mix to each tube.

5. Dilute 10 µ M of ASO solution to 2 µ M .

6. Add 1 µ L of 2 µ M ASO to specified tube, mix well by gently tapping the tube.

7. Incubate the tubes at 37°C for 40 min and heat to 65°C for 10 min to inactivate

the enzyme.