Genomic DNA extraction – Dellaporta mehtod

·         Pros: Speedy, cheap, less dangerous (no phenol or chloroform)

·         Cons: No good with polysaccharides or phenolic complexes, impurities

·         You can expect to get 20 ug from 100 mg of tobacco leaf or 3 ug from 100 mg of Arabidopsis

           1. Materials

·         Extraction buffer (EB)

o    50 mM Tris-HCl, pH 8.0

o    10 mM EDTA, pH 8.0

o    100 mM NaCl

o    1% SDS

o    10 mM beta-mercaptoethanol

o    Liquid nitrogen

o    5 M Potassium acetate, pH 7.5

o    Isopropanol

o    80% EtOH

o    3 M Sodium acetate, pH 5.2

o    65ºC Temperature block or water bath

o    E-tube

o    Falcon 2059 tubes

o    Miracloth (Calbiochem 475855) filters

o    Powder Funnel (15 mm Stem)

o    Spatula

o    pestle and mortar

            2. Procedure

a)     Freeze 1 g of plant material in liquid N2.  Grind to a very fine powder in a cooled mortar and pestle.

v Add liquid N2 to keep material frozen and brittle.

b)    Transfer powder to a 30 ml tube and add 15 ml of extraction buffer.

c)     Add 1 ml 20% SDS and mix thoroughly by shaking.  Incubate tube(s) at 65ºC for 10 min.

d)    Add 5 ml of 5M Potassium acetate and shake.  Incubate tube(s) at 0ºC for at least 20 min.

e)     Spin tube(s) at 25,000xg (14,500 rpm in SS34 rotor) for 20 min.  

f)      Pour supernatant through "Miracloth" (Miracloth can be obtained from Calbiochem Corporation, Lajolla, CA92037, U.S.A.) filter into fresh tube containing 10 ml isopropanol.

g)     Mix and incubate at -20ºC for 30 min.

h)    Spin tube(s) at 20,000xg for 15 min (13,000 rpm in SS34 rotor).  

i)       Pour off supernatant and allow tube(s) to drain by inverting tube(s) on a paper towel.

j)       Redissolve pellet in 0.7 ml of 50mM Tris, 10mM EDTA, pH 8 and transfer to Eppendorf tube(s).  Spin in microfuge for 10 min to pellet insoluble debris.

k)    [Optional...] Transfer supernatant to fresh Eppendorf tube(s), add an equal volume of 1:1 Phenol/Chloroform, mix and spin in microfuge for 10 min.

l)       Transfer aqueous phase to fresh Eppendorf tube(s), add 1/10 volume 3M sodium acetate (75 ul) and 500 ul isopropanol.

m)  Mix well and pellet DNA by brief centrifugation in microfuge.  Wash pellet with 1 ml of ice-cold 80% ethanol.  Dry pellet and redissolve in 100 ul 10mM Tris, 1mM EDTA, pH 8.