CTAB DNA extraction

CTAB DNA extraction kdorfman Wed, 02/24/2016 - 17:21

Materials

Materials kdorfman Wed, 02/24/2016 - 18:28

Equipment

  • microfuge tubes with blue pestles
  • 60C drybath
  • ice
  • microfuge

Chemicals:

Aliquot 600 µL each:

  • CTAB buffer
  • isopropanol (ice cold)

Aliquot 750 µL chloroform:isoamyl alcohol.

More than one/student for repeat extraction

CTAB extraction buffer

Recipe

ingredient concentration amount per L
Tris pH 8 1M 100 mL
NaCl 8.1 % 81.8 g
EDTA 0.5 M 40 mL
CTAB 2% 20 g
Beta ME 0.2% 2 mL

Buy it from Fisher: Teknova C2190 CTAB Extraction Solution:

ingredient concentration
Tris pH 8 100 mM
NaCl 1.4 M
EDTA pH 8 20 mM
CTAB 2%

Add Beta ME just before aliquoting: 2 µL/mL

to make: mL CTAB Extraction Buffer
at this concentration: %
add: µL Beta mercaptoethanol

Student Protocol

Student Protocol kdorfman Wed, 02/24/2016 - 18:27

  • crush leaf tissue in 600 µL 2X CTAB extraction buffer in microfuge tube using blue pestle

  • Incubate 60C 30 min. Invert 2x

  • Add 600 µL chloroform:isoamyl alcohol (24:1)

  • Centrifuge 5 min

  • Harvest supernate. (If it's cloudy, repeat chloroform:isoamyl step)

  • Add equal volume ice cold isopropyl

  • invert gently until 2 phases mix.

  • Store at -20C (at least 30 min, better overnight)