Preparation of G+A Marker

Procedure

Add the following to a sterile microcentrifuge tube:

Labeled target DNA (3-6ng)           1-8ul

Calf Thymus DNA (0.5ug/ul)           2ul

TE buffer                                   0-7ul      

Total Volume                              10ul

Add 1ul of 4% Formic Acid and incubate for 25 min at 37℃

During this incubation, add 15 ul of stock piperidine to 135 ul of water to prepare a 1M piperidine solution.

Place the tube containing the formic acid reaction on ice, add all 150 ul of the diluted (1M) piperidine solution and incubate for 30 min at 90℃.

Place this reaction on ice for 5min, add 1ml of n-butanol and vortex.

Centrifuge for 2min at high speed to pellet the DNA. Remove the supernatant and add 150ul of 1% SDS to the pellet.

Add 1ml of n-butanol, vortex vigorously and centrifuge at high speed for 2 min. Carefully remove the supernatant.

Add 0.5 ml of n-butanol to rinse the pellet, and carefully remove the supernatant. Repeat this rinse step once.

Dry the pellet under vacuum for 10min, adds 5-10ul of loading dye, and mix well. Place at  -20⁰C.until required. This sample may be stored up to two weeks at -20℃.

Note

This protocol was adopted from Amersham footprinting kit instruction.