One Shade Too Dark

--- tale of condensed tannins in faba bean seed coats　

            Cetyl trimethyl ammonium bromide (CTAB) , also known as hexadecyltrimethylammonium bromide or 1-hexadecanaminium, N,N,N-trimethyl-, bromide (C₁₆H₃₃)N(CH₃)₃Br) is one of the components of the antiseptic cetrimide. It is a cationic surfactant. Its uses include providing a buffer solution for the extraction of DNA. (taken from http://en.wikipedia.org/wiki/ Cetyl_trimethylammonium_bromide)

1.   Set water bath to 60°C.

2.   Grind 0.35 g of freeze dried leaf tissue to a fine powder with 3 glass beads using the paint shaker for 9 minutes.

3.   Quickly add 1mL of 2X cTAB buffer (add the B-mercaptoethanol on the day of use) for each 0.35 g and mix again after the mixture has thawed

4.   Incubate for 30 minutes at 60°C in a water bath. Shake every 5 minutes while it is in the water bath.

5.   Sit tube on bench for 10 minutes to allow the mixture to return to room temperature.

6.   Add 1 ml of chloroform:isoamyl alcohol (24:1) to each tube and shake tubes continuously for 5 minutes.

7.   Spin in table top centrifuge for 10 minutes at full speed

8.   Remove the upper (aqueous) phase to a clean tube using a pipette

9.   Add 1 ml of chloroform:isoamyl alcohol (24:1) to each tube and shake tubes continuously for 5 minutes.

10. Spin in table top centrifuge for 10 minutes at full speed

11. Add 1 ml of cold isopropanol and rock tube until DNA precipitates

12. Spin Down DNA for 10 minutes at high speed, decant isopropanol, and add 1.0 ml of 76% ETOH/0.2 M Na-acetate. Leave    DNA in this mixture for 20 minutes.

13. Decant 76% ETOH/0.2 M Na-acetate, and add 0.5 ml 76% ETOH/10 mM NH₄-acetate. Leave here for 5 minutes and then spin for 10 minutes, decant the 76% ETOH/10 mM NH₄-acetate.

2X cTAB buffer

10 mL    100 mM 1 M Tris-HCI pH 8                   

2 g          2 % cTAB                                          

28 ml      700 mM 5 M NaCl                                

4 ml        20 mM 0.5 M EDTA pH 8                     

1 g          1 % Sodium bisulfite (Na₂S₂O₅)     

Add dH₂O until solution equals 100mL

Add on day of use

1 ul    1% B-mercaptoethanol

76% ETOH/0.2 M Na-acetate

Mix 20 ml H₂O and 2.72 g Na-acetate

Add 80 ml 95% ETOH

Add H₂O until 100mL

76% ETOH/10 mM NH₄-acetate

0.077 g NH₄-acetate

80 ml 95% ETOH

Add H₂O until 100m