DNA Isolation for Lyophilized Tissue
From the laboratory of the Institute for Genomic Diversity at Cornell University

o      Harvest fresh, young leaves

o      Freeze in the -80 freezer overnight

o      Dry in the lyophilizer overnight

o      Turn on water bath to 65C

o      Add beta-mercaptoethanol to CTAB buffer and warm this in the water bath

o      Put tissue in the coffee grinder and grind until a fine powder (may take a couple of minutes)

o      Weigh 1.2g (400mg for the 15 ml tube) of ground, lyophilized tissue, into a 50 ml polypropylene centrifuge tube. DNA yields range from 50 to more than 100 ug DNA/100 mg dry tissue.

o      In hood, add 30 ml of warm (65C) CTAB extraction buffer to the ground tissue. (Distribute tissue along the sides of the tube before adding buffer, to avoid clumping of dry tissue along the sides of the tube before adding buffer)

o      Make sure the tubes are closed tightly and mix several times by inversion

o      Incubate in the water bath for 30 min, shaking occasionally.

o      Note:  Chloroform goes through gloves quickly, wear special gloves or change gloves immediately if it comes into contact with you.  Also, use a glass container, chloroform dissolves some plastics.  In hood, add 14 ml of 24:1 Chloroform/IAA.  Rock gently to mix for 5-10 min.

o      Spin in centrifuge for 10 min at 1300-1500g at room temperature

o      Pipette off top aqueous layer to new 50 ml tube containing 30ul of 10 mg/ml RNase A (pre-boiled).  Mix by gentle inversion and incubate for 30 min at room temperature

o      Add 20 ml of isopropanol.  Mix by very gentle inversion.

o      Remove precipitated DNA with glass hook.  If no precipitated DNA is seen, centrifuge

o      Place hooked DNA in 2 ml plastic tube with 1.5ml 70% EtOH

o      Transfer DNA to 2 ml microfuge tube, allow to dry at room temperature for 30 min.  Add 0.6 ml of TE.  Cap tube and rock gently overnight at room temperature to dissolve DNA.  Store samples at 4C.

 

CTAB Extraction Buffer

Stock

[Final]

100 ml

500 ml

1L

ddH20

 

65 ml

325 ml

650 ml

1M Tris 7.5

100 mM

10 ml

50 ml

100 ml

5M NaCl

700 mM

14 ml

70 ml

140 ml

0.5M EDTA, pH 8.0

50 mM

10 ml

50 ml

100 ml

CTAB

1%

1 g

5 g

10 g

β-ME

140 mM

200 ul

5 ml

10 ml