Mini CTAB DNA Extraction Method (Long version for Southern blots)

 

Jacobsen Lab 12/00

1) Wipe off bench top to remove potential contaminating DNA, use 20% Bleach, then water, then ethanol.

2) Set a water bath or heat block to 65C.

3) Add BME to CTAB buffer (20 µl per 10 ml solution) and add 1ml per morter.

4) Grind 3 leaves or more (fresh or frozen) in morter (one gets about 1ug per A thal. leaf).

5) Transfer solution to 1.5 ml eppindorf tube with 1ml pipetman.

6) Incubate .5 - 3 hours at 65C.

7) Spin 5 min high speed in microfuge.

8) Transfer supernatent to new tube containing .75 ml Chloroform/Isoamyl alcohol (24:1). Invert for 2-3 min. Spin 5 min high speed in microfuge.

9) Transfer top layer to a new 1.5ml eppy, being careful not to get the white interface. Add .7 volumns isopropanol (usually one gets about .7 ml of supernatent, so add .49 ml of isopropanol). If less than three leaves were used, also add 20ul (1ug/ul concentration) of glycogen or tRNA. Mix well, by very light vortexing. Spin 15 min high speed in microfuge.

10) Remove as much of the supernatent as possible. Add 200ul of TE. Disolve pellets completely. This is accomplished by light vortexing and or incubation at 37C for several minutes.

11) Add 66ul of 10M NH4OAC. Mix. Add 665ul Ethanol. Mix well. Spin 15 min high speed in microfuge.

12) Remove all of the supernatent. Wash the pellet by adding 1ml of 70% ethanol, inverting the tube once, removing most of the ethanol with a pipetman, spinning the tube very briefly, and removing the rest of the ethanol with a small pipetman. Air dry the pellet for 5 min.

13) Resuspend the pellet in 50ul of TE.

14) To check the quality and quantity of the DNA, run 3 ul on a 0.4% agarose gel.

Add 3 ul DNA sample

1.5 ul RNAse A containing dye.

5.5 ul Water

Incubate 5 min at 37C before loading.

2x CTAB:

Concentration Ingredient For 200 ml:

2 % CTAB 4.0 g

100 mM Tris pH 8 20 ml of 1.0 M soln

20 mM EDTA 16 ml of 0.25 M soln

1.4 M NaCl 16.4 g

2 % PVP 40 4.0 g

0.2% Beta mercaptoethanol Add just before use; 20 µl per 10 ml solution

Notes:

CTAB is Hexadecyltrimethylammonium bromide. Dissolve it before adding salt, with stirring and a little warmth, if necessary.

When the salt is dissolved lots of tiny bubbles come out of solution; they rise to the surface very slowly, simulating undissolved material. Turn off the stirrer 5 minutes after adding the salt, to see if salt is dissolved and only bubbles are present.

PVP of 40,000 average molecular weight makes the solution slightly translucent, but no large particles should be present after dissolving.