DNA extraction

(using the Omega Biotek E.Z.N.A. Mollusc DNA Kit)

Whats needed:
Tissue for extraction, scalpel, wax paper or plastic weighing dish
Heating block set to 60C
Buffer ML1 (from kit)
Buffer ML2 (from kit)
Proteinase K (from kit)
Chloroform:isoamyl alcohol (or just chloroform)
Absolute ethanol (95-100%)
Isopropanol
HiBind DNA column (from kit)
2 ml collection tube (from kit)
DNA Wash Buffer (make sure the EtOH has been added)
1.5 ml centrifuge tube
Elution Buffer (from kit)
Useful tips:
This is a several hour to two day procedure depending on length of incubation needed to completely
break down tissue.
Multiple samples can be done at once; label tubes accordingly (e.g., keep same numbers for different sets
of tubes.
Label all tubes prior to use.

PROCEDURE (modified from Omega Bioteks protocol)

[Steps 1-5 can be done in approximately 1 hr depending on the number of samples being extracted.]
1.

Place 350 ul of Buffer ML1 into a numbered 1.8 ml centrifuge tube.

2.

Remove approximately 25 mg of tissue from specimen (no more than about 5 mm3 of
tissue). Mince tissue by making cross cuts. Do dissections and cross cuts on wax paper
or in a plastic weighing dish.

3.

Place tissue in tube with the 350 ul Buffer ML1.

4.

Add 25 ul Proteinase K (~20 mg/ml) to tube.

5.

Incubate tube at 60C for at least 30 minutes or until majority of tissue is dissolved.
[Overnight incubations can be done 60C or 37C.]

[This is a good stopping point, incubation can be run from 30 minutes to several hours or even
overnight if needed.]
[The remaining steps shouldnt take more than about 1 hr.]
6.

Add 350 ul chloroform:isoamyl alcohol (or chlorofrorm) to tube. Gently mix

solution in tube by inverting tube (up and down) 5-10 times. This step should be done in
the fume hood.

7.

Centrifuge tube at 10,000 g for 5 minutes.

8.

Carefully remove upper aqueous layer from tube and place in a new numbered 1.8 ml
tube. Avoid taking any of the whitish interphase at the bottom of the
aqueous layer even if this means not being able to remove all of the
upper phase. [If there is not much of an aqueous layer or the aqueous and organic phases
do not separate well, add about 200 ul of Buffer ML1 to tube, mix and centrifuge again.]
[Discard organic phase in appropriate container and discard all tubes and tips that came into
contact with the chloroform in appropriate contaminated plastics bin.]

9.

Use a pipette to estimate the volume of the transferred aqueous phase.

10.

Add 0.7 x volume of Isopropanol to tube. (e.g., if you have 350 ul of the aqueous
phase, then add 245 ul (350 ul x 0.7) of Isopropanol). Gently mix solution in tube by
inverting tube (up and down) 5-10 times. Look for spooled DNA that will look like a
small white, cloudlike substance -- having spooled DNA is a good sign of having high
quality DNA, but not having it is ok too!

11.

Incubate solution at room temperature for 10 minutes on bench.

12.

Centrifuge tube at 10,000 g for 10 min.

13.

Look for pellet of DNA at bottom of tube and carefully discard the supernatant without
disturbing pellet.

14.

Air dry pellet by keeping tube open on bench top for 2-5 min. Use SpeedVac if you want
to get the pellet completely dry, but do not use heat (or pellet can get glued to the tube!).

15.

Resuspend pellet in 300 ul sterile water (PCR water). Flick tube to dislodge pellet from
wall of tube and make sure all of pellet dissolves. [If necessary, incubate tube at 60C until
pellet dissolves.]

16.

Add 200 ul Buffer ML2 and 300 ul absolute ethanol (95-100%) to tube.

17.

Place a HiBind DNA column into a 2 ml collection tube.

18.

Place all of the solution from tube into the HiBind DNA column.

19.

Centrifuge column with collection tube at 10,000 g for 1 min.

20.

Place the column into a new 2 ml collection tube.

21.

Do first wash: Add 700 ul DNA Wash Buffer (make sure ethanol was added) to
column.

22.

Centrifuge column with collection tube at 10,000 g for 1 min.

23.

Discard flow through from collection tube.

24.

Do second wash: Add 700 ul DNA Wash Buffer (make sure ethanol was added) to
column.

25.

Centrifuge column with collection tube at 15,000 g for 2 min (note longer spin time than
above!).

26.

Place column into a new, numbered (or labeled; this is the final sample tube!) 1.5 ml
centrifuge tube.

27.

Add 30 ul of preheated (to 60-70C) Elution Buffer (or 10 mM Tris buffer, pH 9.0)
to column.

28.

Let soak for approximately 2 min at room temperature.

29.

Centrifuge column with tube at 10,000 g for 1 minute.

30.

Label tube and store at -20C (or use now as template for pcr!).

The final tube has your final genomic DNA (gDNA) extract. To check quality/quantity of gDNA
you can run 2 ul of solution (with 2 ul of loading dye) on a 2% agarose/0.5X TBE gel (or do an
amplification and check to see if it can be pcrd from gDNA can also be run on the gel used to assay
the pcr). Use should be able to use 1 ul of solution as the template for pcr; if the DNA is very
concentrated, you will need to dilute the gDNA about 10-100 fold.