1. Prepare the TLC plates by warming it in the oven for a few minutes
2. Dry the samples under N2 at 60oC
3. Make a 2:1 solution of chloroform to methanol
4. Obtain the TLC plates and draw the lines
5. Mark a lane for the SPC standard on each plate
6. Load 20uL of SPC to the SPC lane on each plate
7. Obtain each sample and load 50ul of 2:1 solution to the tube.
8. Dissolve the particles and rinse the edges of the tubes to dissolve any lipids
9. Add 10uL spc to the 50ul already added
10. Mix the solution and then load
11. Rinse out any remaining lipid by loading 25uL of the 2:1 solution into the tube and then load
12. Repeat this process for each sample
13. Place into a chamber of solvent make of CHCL₃ (65mls): MeOH (25mLs): H₂O (4mls), until the solvent line reaches the top
14. Remove the plate and place into a 70oC oven for a few minutes
15. Place the plate into an iodine chamber until the bands appear.
16. Pencil in a cm above and below the band and let the plate sit for a few minutes until the bands fade
17. Obtain scintillation vials and load 4mls of the ecolite solution
18. Scratch out the band area and load the powder into the properly labeled vial.
19. Place the sample into the beta counter for the samples to be read.

 

TO Make SPC Marker: (1mg of SPC + 1mL of Chloroform:Methanol)

Ex. To make 6.5mg

       Weigh out 0.00657g

      + 3.3mLs Chloroform

       + 3.3mLs Methanol

 

TO correct data using DNA Assay Method:

Making DPA Solution                                 Note:

-          Protocol Recipe                                Use only the amount needed

-          75mL Glacial Acetic Acid                      (adjust volume)

-          1.123g of Diphenylamine (DPA)                   DPA solution Is light

-          1.125mL of H2SO4                                sensitive and will not

-          375uL of acetaldehyde                           last for more than a few days

 

Making DPA solution from 37.5mLs of glacial

37.5mLs GAc + 0.5625g DPA + 0.5625mL H2SO4 + 187.5uL acetaldehyde

 

DPA solution by taking 1/3 of the total volume listed in the protocol recipe

25mls GAc + 0.375g DPA + 375uL H2SO4 + 125uL acetaldehyde

45:0.674:674:225

40:0.6:600:200

50:0.45:450:150

11:0.165:165uL:55uL

 

 

 

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