1. Cold KI-I2 solution: 3.95g of I2 and 5g of KI are dissolved in 25ml of water. The mixture is shaken for 45 min and stored at 4C (sealed w/ parafilm)
2. Ethylene dichloride
3. 4N NH4OH
4. Cold 1N H2SO4
5. Dowex-1 (OH-) : Dowex-1 (Cl-) -> washed w/ 6M NaOH -> rinsed w/ water (>5 times)

Methods:

1. Treated cells are rinsed with dd water three times.
2. Freeze-dry overnight
3. Weigh 50-100mg of samples
4. Extract with 5ml of methanol at 4C for 24h (or 5ml of methanol containing 0, 50, 100, 300, 600 ug betaine standard)
5. Add 2.5 ml of chloroform and shake for 30sec
6. Add 3 ml of dd water and vigourly shake for 10-15min
7. Stand for 1-2 hours until clear phases appear
8. Remove the upper aqueous phase (~7ml) and evaporate into dryness
9. Re-dissolve in 2 ml of water
10. Add 0.5ml of Dowex-1(OH-) resins (2 vol resin : 1 vol water) and shake for 10 min
11. Transfer supernatant to new vials and also collect 2 x 1 ml of water-elutant
12. Add 0.5ml of Dowex-50(H+) resins (2 vol resin : 1 vol water) to elutant and shake for 1h
13. Discard supernatant and wash resins with 2 x 2 ml of water
14. Elute with 2 x 2 ml and then 1 x 1ml of 4N NH4OH
15. Evaporate eluant to dryness
16. Re-dissolve in 0.6 ml of 1N cold H2SO4 and transfer 0.5 ml into 1ml eppendorf tubes
17. Cool to 4C
18. Add 0.2 ml of KI-I2 reagent, tap the tubes gently, and centrifuge
19. Stand at 4C overnight or at least 6h
20. Centrifuge at 14,000rpm at 4C for 30 sec
21. Aspirate the supernatant using a Pasteur pipette with a very fine tip (pull your own)
22. Rinse pellet with 2 x 1 ml cold 1N H2SO4
23. Centrifuge at 4C and aspirate the supernatant
24. Dissolve periodide pellet with 0.8 ml of ethylene dichloride
25. Dilute 25ul of periodide samples with 2.5 ml of ethylene dichloride
26. Read at 365 nm with Beckman DU-7 spectrophotometer

References:
1. Wall et al. (1960) Spectrophotometric determination of betaines and other quaternary nitrogen compounds as their periodides. Analytical Chemistry 32(7): 870-874
2. Ladyman et al. (1983) Genotypic variation for glycine betaine accumulation by cultivated and wild barley in relation to water stress. Crop Science 23: 465-468
3. Rhodes et al. (1987) Determination of betaines by fast atom bomardment mass spectrometry. Plant Physiol. 84: 781-788