GDNA extraction using CTAB Buffer

From ChoanoWiki

2 x CTAB DNA Isolation Procedure for Choanoflagellates

This method is useful for difficult samples, for example where contaminants are interfering with PCR processes

Recipe for 2x CTAB buffer

• 10 ml 1M Tris Hcl pH 8
• 35 ml 4M NaCl
• 4 ml 0.5 EDTA pH 8
• 2 g CTAB
• dd Water to 100 ml
• Autoclave, then add 200 µl of 2 ß-mercaptoethanol

Protocol for DNA extraction

1. Pellet cells by spinning at 2700g for 40min at 4^oC

Carefully remove the supernatant

Add 600 µl of 2x CTAB buffer and vortex to resuspend the cells

Add 10 µl Proteinase K to each tube

Incubate samples at 55 °C until tissue is digested (a 5 hour incubation works for loricate choanoflagellates)

2. Add 600 µl of ice cold chloroform to each tube

Mix tubes thoroughly

Centrifuge at 4 °C for 15 minutes at 13000rpm

3. Transfer the upper layer to a new tube and repeat step 2 until the interface between the layers is clean and free of any white precipitate (only usually need one repeat).

4. Transfer the clean upper layer to a new tube and add 600 µl of ice cold isopropanol.

Invert tube several times (gently) and watch the DNA precipitate into strands.

Centrifuge at 13000g for 10 minutes to pellet the DNA

5. Carefully use a pipette to draw off the supernatant

6. Wash pellet in 800 µl of 70% ethanol

Centrifuge at 13000g for 10 minutes to precipitate the DNA

Carefully remove the supernatant ethanol

7. Allow pellet to dry completely and resuspend in 30µl of dd water

Footnote: Protocol posted by Alan Marron July 2009