Calculations:

75 x 1/3 = 25mLs GAc

1.125 x 1/3 = 0.375g DPA

1.125mL H2SO4 x 1/3 = 0.375mls H2so4 x 10-3L/mL x 1uL/10-6L = 375uL h2so4

375uL x 1/3 = 125uL acetaldehyde

 

DNA Analysis

1. Prepare Standards:

400nM DNA (uL)              DNA (ug)                   H2O

0                                  0                         50

12.5                             5                         37.5

25                                10                       25

50                                20                       0

2. Add perchloric acid to each tube (samples and standards) (250uL)
3. Put in oven at 70C for 15 min. Cool.
4. Make DPA reagent: 75mL GAc

       1.125g DPA

        1.125mL H2SO4

        Add 375 uL acetaldehyde to DPA reagent

5. Add 1mL of this solution to each tube, vortex
6. Parafilm samples and leave for 24 hours to allow for shades of blue color to develop
7. Read samples in the spec at 600nm
8. Calculate Dna Concentrations

DNA Standards: 400nmoles/mL: 2 deoxy-L-ribose in 0.01M PBS (ph 6.0) = (400nmoles = 0.04mg/ml)

Acetaldehyde = 16mg/mL of water

 

To correct using protein assay follow glass tube method.

 

Triolein Solvent Recipe:

Petroleum Ether = 112.5mL; Ether = 36mL; GAc = 1.5mL

Note: Only changes made are the solvent and the marker for triolein

 

Choline Solvent Recipe:

Chcl3 = 97.5mL ; meoh = 37.5mL; water = 6mL

 

 

Post-choline Labeling Assay of HL60 Cells  

[3H] Choline Labeling Assay of HL-60 Cells  

 

 

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