Jimenez-gomez lab:protocol CTAB 96 well DNA extraction
(Redirected from JMJG Lab:protocol CTAB 96 well DNA extraction)
96-Well Format DNA Extraction with CTAB
- Add a single ball bearing to each 1.2 ml tube containing tissue. (This can be done by using a sealing mat with 96 indentations and sprinkling the ball bearings over the top). Put samples into liquid nitrogen.
- Disrupt tissue by shaking with paint shaker for 2 min. Repeat if clumps of tissue remain.
- Centrifuge briefly to bring down tissue dust.
- Add 300 ul CTAB, place sealing mat loosely on top of plate and heat at 65°C in water bath for 30 min. (optional)
- Let cool to room temperature.
- Centrifuge briefly and add 300 ul chloroform (done in hood). Use a TIP BOX lid for chloroform; it melts the boats! Seal tightly with the sealing mat and vortex vigorously for 10-20 seconds. Or use the mix step on the Apricot.
- Centrifuge at 3250 rpm for 15 min.
- During centrifugation, prepare 96-well deep well plates by adding 200 ul isopropyl alcohol to each well.
- Transfer 200 μl of the chloroform-extracted supernatant to the new plates. Be very careful not to transfer any of the goop from the interface, or any of the organic layer.
- Cover the tubes with the sealing mat and centrifuge at 3250 rpm for 15 min.
- Pour off the liquid into the sink; the pellet of DNA should stay behind.
- Wash with 200 μl of 70% ethanol, re-cover the tubes and centrifuge for 7-10 min. at 3250 rpm.
- Dump off the liquid again and blot the plates dry on paper towels. Cover the plates with a kimwipe and let them air dry for >3 hours (or until no liquid can be seen inside and the tubes don’t smell like ethanol).
- Resuspend the pellets in 100 ul of TE and let sit for >3 hours at 4°C (I usually leave them overnight). The DNA can then be transferred to skirted 96-well PCR plates for storage.
- Use 0.5 μl in a 10 μl PCR reaction.
1 Liter of 2X CTAB
- 20g CTAB (cetyltrimethyl or hexyltrimethyl ammonium bromide)
- 81.82g NaCl
- 100 ml 1M Tris, pH 8
- 40 ml 0.5M EDTA
- (CTAB goes into solution slowly and can release toxic fumes if heated)
- Final cc:
- 2% CTAB, 1.4M NaCl, 100 mM Tris, 20mM EDTA