CTAB genomic DNA extraction from Arabidopsis leaf material
Diep R Ganguly, Pip Wilson, Gonzalo Estavillo, Xin Hou, Barry Pogson
Abstract
CTAB-based extraction of genomic DNA from Arabidopsis leaf tissue.
Before start
Ensure you grind your leaf tissue into a fine powder using mortar and pestle or Qiagen tissue lyser (place 1/8" steel ball bearing into tube with tissue sample).
Make sure leaf tissue remains frozen until the addition of CTAB buffer.
Steps
Cell lysis
Prepare 2% CTAB buffer.
| A | B |
|---|---|
| Reagent | [Cf] |
| hexadecyltrimethylammonium bromide | 2% (w/v) |
| NaCl | 1.4 M |
| EDTA (pH 8) | 20 mM |
| Tris-Cl (pH 8) | 100 mM |
2% CTAB buffer recipe
Aliquot required volume of CTAB buffer and heat in water bath at 60 ºC for 5-10 minutes immediately before use.
Add 300 µL / 100 mg leaf tissue of CTAB buffer.
Add RNase A solution to a concentration 50-100 µg/mL.
Mix well with a vortex. Invert samples by hand to ensure that all ground tissue is in solution.
Incubate in water bath at 60 ºC for 30 - 60 minutes. Mix tubes periodically by inversion.
Cool samples to room temperature .
Phase separation
Add 300 µL chloroform and mix thoroughly with a vortex or vigorous shaking for 15 seconds.
Centrifuge samples for 10 minutes at 20,000 rcf.
Transfer upper aqueous (approx. 200 µL) phase to clean tube.
Repeat steps 9-10 for a cleaner extract.
Precipitation
Add equal volume of ice-cold 2-propanol and mix by inversion.
Incubate for 30-60 min @ -20 °C
Centrifuge for 15 min @ 20,000 rcf.
Discard supernatant using pipette.
Resuspend DNA
Wash pellet with 1 mL of 70 % ethanol (mix by inversion).
Centrifuge samples @ 9,200 rcf for 5 min.
Remove as much ethanol as possible using a pipette, then allow pellet to air dry for 5 minutes.
Resuspend gDNA in nuclease-free H2O or low EDTA TE buffer (10 mM Tris-Cl pH 8, 0.1 mM EDTA).
Test yield and purity of samples using a Nanodrop and running samples on a 1 % agarose gel.
