Based on the Shaw Lab protocol (S.B.B 11/15/96) and recommendations from Blanka Aguero

Process

Preparation

  1. Sign up for the Genogrinder.
  2. Add 2 stainless steel ball bearings to each racked 1.2ml “collection” tube.
  3. Sample a small amount of the capitulum into tubes, pushing the tissue into the bottom of the tube. Save the remainder of the plant in a mini-packet and leave with the herbarium specimen as a voucher.
  4. Turn on water bath (60–65C), and place isopropanol and 70% ethanol in -20C freezer to cool.
  5. Start preparing fresh CTAB isolation buffer. We will add the 2X stock CTAB buffer and PVP-40 now and add the B-mercaptoethanol just before using. Make it in 15uL or 50uL falcon tubes. Add the PVP-40 first, then the 2X stock CTAB buffer. Mix with a spatula to break up large chunks of PVP-40 and place the tube in the water bath to fully dissolve while grinding tissue. Make enough extra to allow using the multipipetter:
    • Reagent per tube per 48 samples (+6 extra) per 96 samples (+12 extra)
      2X stock CTAB buffer 500uL 27mL 54uL
      PVP-40 0.02g 1.08g 2.16g
      B-mercaptoethanol 2uL 108uL 216uL

Grinding tissue

  1. Gather:
    • The small blue dewar
    • Autoclave/liquid-nitrogen gloves
    • Old forceps
    • A small shallow cooler
    • Samples in balanced racks with holey bottoms
  2. Get 0.5L of liquid nitrogen (BioSci 233). You will need a purchasing code for the log.
  3. In the Willis lab (French 3332), freeze the tissue by dipping the racked tubes in a cooler containing a small amount of liquid nitrogen (~2 cm in depth). Hold the rack with the forceps. Freeze for 5-10 seconds.
  4. Transfer balanced racks immediately to the Genogrinder and construct the “sandwich” with the top rubber gasket directly against the caps of the tubes (i.e. no box lid!!). Grind at the 1X setting, speed 500 for 1-1/2 minutes. Check that tissue is fully ground–regrind as needed.

Extracting with CTAB isolation buffer

  1. Back in the Shaw lab, in the hood, add B-mercaptoethanol to the warm CTAB buffer+PVP40 to complete the CTAB isolation buffer.
  2. Add 500µl CTAB isolation buffer to each sample. You can use the 1250 multichannel pipette for this.
  3. Hold caps closed and vortex well to get the powdered tissue mixed with the extraction buffer. Incubate the crude extract in a 60C water bath for 60 min. Place weighted lid on top of capped tubes to prevent opening during the incubation. Hold caps closed and gently mix by inverting every 20-30 minutes during the incubation.
  4. During the 60 min incubation, prepare 200uL wide-cut tips, label a new set of collection tubes, and prepare 24:1 Chloroform:Isoamyl alcohol.
  5. Remove tubes from hot water bath without dislodging the weight holding the caps on. Blot any moisture from the tops of the tubes.
  6. Turn off hot water bath.

Removal of proteins with chloroform:isoamyl alcohol

  1. Add an equal volume (500µl) of chloroform:IAA (24:1) to plant extract. You can do this with the 1250 multichannel pipette, but use a glass dish–the mixture will dissolve plastic troughs.
  2. Hold caps closed and mix gently by inversion (30-50x) to produce an emulsion.
  3. Centrifuge balanced racked tubes for 5 min. at 4500 rpm in the plate centrifuge to separate the phases.
  4. Remove and save the aqueous (top) phase to new labeled collection tubes using a p200 pipetter with wide-cut tips to reduce DNA shearing. Avoid the interface! Estimate volume of the aqueous phase to the nearest 50uL (~400uL typical).

Precipitation, washing, and drying of DNA

  1. Add an equal volume (400uL typical) of cold isopropanol to aqueous phase.
  2. Mix gently by inversion.
  3. Precipitate 30 min. at -20C. Longer precipitation leads to more impurities.
  4. Centrifuge for 20 min. at 4500 rpm in the plate centrifuge. Pour off and discard supernatant.
  5. Wash pellet with 500µl cold 70% ethanol (pellet will not resuspend). Spin 5 min. at 4500 rpm to secure pellet to tube side. Carefully pour off and discard supernatant. Repeat to wash pellet a second and third time.
  6. Cover open tubes with a kimwipe and air dry overnight (or dry ~10 min at room temperature in Speed-vac, if available).
  7. Resuspend pellet in 25uL water for several hours to overnight in the fridge before measuring concentration. (Optional: incubate for 5 min. at 60C to assist with resuspension.)

Recipes

2X stock CTAB buffer:

  • 10mL 1M pH8 Tris
  • 8.2g NaCl
  • 4mL 0.5M EDTA pH8
  • 2g CTAB
  • water to 100mL

Stir on warm hotplate (don’t boil) until CTAB is in solution. Store at room temperature and use or discard within 6 months.

1M pH8 Tris:

  • 121.1g Tris
  • 700mL water
  • Dissolve tris and bring to 900mL
  • pH to 8 with concentrated HCl (will need ~50mL)
  • water to 1L

0.5M pH8 EDTA:

  • 46.5g EDTA disodium salt dihydrate (ONLY!)
  • 200mL water
  • pH to 8 with NaOH pellets (will need 2.5–4g. EDTA will not dissolve until pH is near 8. Even then it takes time—gentle heat may help.)
  • water to 250mL