Genomic DNA Isolation

1. Weigh 300-400 mg of ground, lyophilized tissue, into a 15 ml polypropylene centrifuge tube. DNA yields
range from 50 to more than 100 μg DNA/ 100 mg dry tissue.
If higher amounts are needed, start with 1 g lyophilized tissue into a 50 ml polypropylene centrifuge tube, and
triple all the amounts given below. If lower amounts are needed, then weigh 100-150 mg lyophilized tissue
into a 5 ml polypropylene centrifuge tube, and use 1/3 of the amounts given below.
2. Add 9.0 ml of warm (65•C) CTAB extraction buffer to the 300-400 mg ground, lyophilized tissue. It is best to
distribute tissue along the sides of the tube before adding buffer, to avoid clumping of dry tissue in the bottom.
Mix several times by gentle inversion.
3. Incubate for 60-90 min, with continuous gentle rocking in a 65•C oven.
4. Remove tubes from oven, wait 4-5 min for tubes to cool down, and then add 4.5 ml chloroform/octanol (24:1).
Rock gently to mix for 5 -10 min.
5. Spin in a table-top centrifuge for 10 min at 1300-1500 x g1 at RT.
NOTE:

 below 15•C the CTAB/nucleic acid complex may precipitate; this could ruin the preparation and
cause damage to the centrifuge.
6. Pour off top aqueous layer into new 15 ml tubes. Add 4.5 ml chloroform/octanol and rock gently for 5-10 min.
7. Spin in a table-top centrifuge for 10 min at 1300-1500 x g1 at RT.
8. Pipette off top aqueous layer into new 15 ml tubes containing 30 μl of 10 mg/ml RNase A (pre-boiled). Mix by
gentle inversion and incubate for 30 min at RT.
9. Add 6.0 ml of isopropanol (2-propanol). Mix by very gentle inversion.
10. Remove precipitated DNA with glass hook2. Continue with OPTIONS A, B, or C.
OPTION A: phenol extraction, to obtain DNA of higher purity
NOTES:

this option is highly recommended for DNA analyses based on the polymerase chain reaction
(PCR), such as RAPDs.
For RFLP analyses, this option is usually not necessary unless DNA does not digest properly. In
fact, it is better to perform a phenol extraction only after restriction digestion; this improves DNA
band separation and resolution after electrophoresis (see later sections for details).
10. Place hook with DNA in 5 ml plastic tube containing 1 ml of TE; gently twirl hook until DNA slides off the
hook. Cap tubes and rock gently overnight at room temperature to dissolve DNA.
11. Phenol extract each sample with 1 ml (1x original TE volume) of equilibrated phenol or 1:1
phenol:chloroform. Centrifuge the sample 10 min at 1300 x g1 in swinging bucket rotor.
12. Transfer top (aqueous) layer to new 5 ml tube. Extract DNA with a 1 ml (1x original TE volume) of
chloroform/octanol . Centrifuge the sample 10 min at 1300 x g1 in swinging bucket rotor. Transfer top
(aqueous) layer to new 5 ml tube. Continue with step 14 of OPTION B.

OPTION B: ethanol precipitation
13. Place hook with DNA in 5 ml plastic tube containing 1 ml of TE; gently twirl hook until DNA slides off the
hook. Cap tubes and rock gently overnight at room temperature to dissolve DNA.
14. Precipitate DNA by adding 50 μl of 5 M NaCl and then 2.5 ml absolute EtOH (2.5 original TE volume), mix
by gentle inversion.
15. Remove precipitated DNA with glass hook. Continue with step 16 of OPTION C.
OPTION C: DNA washes
16. Place hook with DNA in 5 ml plastic tube containing 3-4 ml of WASH 1. Leave DNA on hook in tube for
about 20 min.
17. Rinse DNA on hook briefly in 1-2 ml of WASH 2 and transfer DNA to 2 ml microfuge tube (preferably
Sarsted with screw-on lids to avoid possible evaporation of the TE) containing 0.3-1.0 ml TE (based on our
experience we use 0.3-0.5 ml for maize and 0.5-1.0 ml for wheat); gently twirl hook until DNA slides off the
hook. Cap tube and rock gently overnight at room temperature to dissolve DNA. Store samples at 4•C.
CTAB Extraction Buffer1.

                                                                   1 RXN                  5 RXN               10 RXN                    20 RXN                 50 RXN                     60 RXN

STOCK                          [FINAL]           10 ml                   50 ml                 100 ml                    200 ml                 500 ml                       600 ml
dH2O                                                        6.5 ml                 32.5 ml              65.0 ml                  130.0 ml              325.0 ml                   390.0 ml
1 M Tris-7.5              100 mM           1.0 ml                 5.0 ml                 10.0 ml                  20.0 ml                50.0 ml                      60.0 ml
5 M NaCl                    700 mM            1.4 ml                 7.0 ml                 14.0 ml                  28.0 ml                 70.0 ml                     84.0 ml
0.5 M EDTA-8.0     50 mM               1.0 ml                 5.0 ml                 10.0 ml                  20.0 ml                50.0 ml                      60.0 ml
CTAB2                        1 %                        0.1 g                   0.5 g                     1.0 g                        2.0 g                      5.0 g                            6.0 g
14 M BME 3               140 mM            0.1 ml                0.5 ml                   1.0 ml                     2.0 ml                   5.0 ml                         6.0 ml

1 Use freshly made; warm buffer to 60-65•C before adding the CTAB and BME.
2 CTAB = Mixed alkyltrimethyl-ammonium bromide (Sigma M-7635)
3 Add BME (•-mercaptoethanol) just prior to use, under a fume hood.

WASH 1:      76% EtOH, 0.2 M NaOAc
STOCK                                  100 ml                               200 ml                             300 ml                           400 ml                              500 ml
Absolute EtOH                   76 ml                                152 ml                              228 ml                            304 ml                              380 ml
2.5 M NaOAc                       8 ml                                    16 ml                                  24 ml                               32 ml                                 40 ml

dH2O                                      16 ml                                  32 ml                                  48 ml                               64 ml                                 80 ml

WASH 2:      76% EtOH, 10 mM NH4OAc
STOCK                                   100 ml                               200 ml                              300 ml                             400 ml                            500 ml
Absolute EtOH                     76 ml                                152 ml                               228 ml                             304 ml                             380 ml
1 M NH4OAc                            1 ml                                      2 ml                                    3 ml                                  4 ml                                   5 ml
dH2O                                        23 ml                                   46 ml                                  69 ml                               92 ml                               115 ml

CHLOROFORM:OCTANOL: 24:1
STOCK                                    100 ml                               200 ml                                300 ml                            400 ml                              500 ml
Chloroform                             96 ml                                192 ml                                288 ml                             384 ml                               480 ml
Octanol                                        4 ml                                     8 ml                                   12 ml                                16 ml                                  20 ml

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