Rumex optimized sorbitol CTAB DNA extraction - SIWLab/Lab_Info GitHub Wiki

Wright/Barett Lab: DNA Extraction Procedure - Version 4 05/03/2018

Solutions (adapted from Benchmarks (1994)):

Pre-treatment buffer (50mL): 250mM sorbitol = 2.275g 25mM Tris-HCl = 1.25mL 1M Tris buffer stock 10mM EDTA = 0.29g or 1mL 1M buffer stock 20mM sodium sulfite = 0.105g (cd try sodium bisulfite if we buy some) 0.1% BME = 1uL / mL, added just before beginning extraction

CTAB lysis buffer (50mL): 100mM Tris-HCl: 5mL 1M buffer stock 1.4M NaCl = 4.09g 20mM EDTA = 0.29g 2% CTAB = 1g 4% PVP = 2g 2% BME = 20uL / mL, added just before beginning extraction

Procedure (Adapted from Storchova et al. (2000)):

Just prior to beginning, add BME to sorbitol buffer and CTAB buffer. Keep 2-mercaptoethanol in the hood. You will need 1.8mL sorbitol pre-treatment buffer and 500uL CTAB buffer for every sample.

  1. Place about .2g of fresh leaf tissue into a round-bottomed 1.5mL centrifuge tube. Put the tube into liquid nitrogen for 5 min. Pre-grind the frozen tissue with a pipette tip before homogenizing with steel/glass balls in the genogrinder. This step is crucial, must be done quickly, and tissue not allowed to thaw. Tissue should be powdery in texture.

  2. Add 1.3mL of pre-treatment buffer is added to each tube.

  3. Mix well by hand and leave suspension for 30 min on ice, periodically inverting.

  4. Centrifuge for 8 min at max speed.

  5. Remove and discard the supernatant carefully (pellet will not be solid, nor will it adhere to the bottom of the tube). Add 300uL pre-treatment buffer, then 400uL lysis buffer. Recap, mix well and place in pre-heated 65C bath. Incubate for 30 min. Occasional mixing throughout/prolonged incubation will increase yield.

  6. Remove samples from bath, noting duration they were heated. Allow samples to cool to room T (5 min).

  7. In the hood, add 600uL CHCL3:isoamyl 24:1, cap tightly and mix very well. Centrifuge 5 min at 8’000 rpm.

  8. Remove 600uL of aqueous phase to a fresh 1.5mL tube. Make certain not to transfer any organic phase. Place CHCl3-containing tubes in the hood for disposal.

  9. To the aqueous phase, add 400uL isopropanol. Mix very well by repeated inversion. Place in -20C freezer for 15+ min.

  10. Remove from freezer and centrifuge for 10 min at max speed. Discard supernatant. Pellet should be stuck to tube wall, so you may pout the supernatant into a waste beaker.

  11. Add 500uL 70%EtOH, mix by hand and centrifuge 5 min at max speed.

  12. Carefully discard supernatant by pipetting.

  13. Place tubes with caps open on the dry bath set to 50C.

  14. When dry, dissolve DNA in 40uL 1X TE buffer, mix well and spin down. Store in fridge/freezer.