Preparation of G+A Marker

Preparation of G+A Marker 
　　Author: Long-Cheng Li 
　　Source: Protocol Online 
　　Abstract: Simplified method for preparing G+A ladder run along with footprinting reaction. It's much 　　　　　　　simple than the original Maxima-Gilbert sequencing reaction and works fine. 

Procedure 　

1、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

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

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

4、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℃.

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

6、Centrifuge for 2mi8n at high speed to pellet the DNA. Remove the supernatant and add 150ul of 1% SDS to the pellet.

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

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

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

This protocol was adopted from Amersham footprinting kit instruction