CTAB DNA Extraction
Prep:
-Get liquid nitrogen
-Turn on heat block to 55 C (immediately after collecting tissue)
-Turn on speed vac concentrator (approx. 1 hr before use)
1. Collect a small piece of young, fresh leaf tissue (~0.5 cm2). Thoroughly freeze by
immersing in liquid nitrogen.
2. Make aliquot of CTAB buffer (500 ul/sample and add β-Mercaptoethanol (7 ul/1
mL CTAB)
3. Thoroughly grind tissue with a plastic pestle and resuspend in 500 ul CTAB lysis
buffer. (RNAse can be added at this step to remove RNA if desired- add 4 ul.)
4. Vortex and incubate at 55 C for 30 minutes – 1 hour.
5. OPTIONAL CLEANING STEP: If desired/necessary, a phenol extraction step can be
performed as follows. Add 500 ul of phenol:chloroform:isoamyl alcohol (25:24:1).
Mix by shaking. Centrifuge at max speed for 10 minutes in bench top
microcentrifuge (room temperature). Pipet off top (aqueous) phase and transfer
to new eppendorf tube.
6. Add 500 ul of chloroform:isoamyl alcohol (24:1). Mix by shaking (if genomic
shearing is a big problem, be more gentle at mixing steps). Centrifuge at max
speed for 10 minutes in bench top microcentrifuge (room temperature).
7. Pipet off the top (aqueous) phase and transfer to a new eppendorf tube
containing 500 ul chloroform:isoamyl alcohol (24:1). Mix by shaking. Centrifuge
at max speed for 10 minutes in bench top microcentrifuge (room temperature).
8. Pipet 375 ul from the top (aqueous) phase and transfer to a new eppendorf tube
containing 250 ul cold isopropanol. Make sure to not transfer any of the organic
layer. If you have to take less than 375 ul, do so. Mix by inversion. Centrifuge at
max speed for 10 minutes in bench top microcentrifuge (4 C).
9. Pipet off supernatant (OR aspirate off with vacuum line). Wash pellets with 250 ul
cold 70% ethanol. Centrifuge at max speed for 5 minutes in bench top
microcentrifuge (4 C).
10. Pipet off supernatant (OR aspirate off with vacuum line). Dry pellets for 5-10
minutes in speed vac (no heat) to remove residual ethanol.
11. Dissolve pellets in 50 ul ddH20 or TE (10 mM Tris-HCl; 1 mM EDTA). Let sit for 510 minutes.
12. Store at -20 C.
Note: For standard PCR use 1 ul of 10-100 fold dilution of DNA stock.