KS USDA, St. AmandRevised on 19-Oct-2014

High Throughput DNA Isolation From Leaves Using SDS& 2mM TCEP in Plates

Based on the Sommers lab method. 2-4 leaf pieces, very young tissue, 4 cm long each. (For oven drying tissue, 35°C to 40°C, 2 to 4 days in oven, 50°C is TOO HOT.Today's date: ______.

1. Grind freeze-dried leaf tissue in the Mixer Mill for 2-5 min at 30/sec using one 4.5 mm stainless steel bead in a labeled 96 deep well plate (1 ml/well).

2. NOTE PLATE NAMES & MISSING WELLS AND RECORD ALL.

3. Use robot to add 450 µl isopropanol to new, labeled, empty 1 ml plates. Put into -20°C, mats are optional.

4. Add 600ml EB to robot stir plate, set speed to 4.5 and allow time for mixing. Use robot with wide-bore P200 tips to add 560 µlRT EBinto each sample well.Cap the plate withmats, roll mats on tightly, place plates in grinder and wet-grind at 30 cycles/sec for 5 sec. Flip plates (so that outer edge is now the inner edge) and wet-grind at 30 cycles/sec for 5 sec. Do not exceed 10 sec total. Remove mat and set aside for re-use, wipe top, place in 65°C oven for 60 min. Recap plates, clamp and invert 5X, then immediately centrifuge samples (4,000 rpm, 3,000g) for 1 SECOND (to settle the bubbles).

5. Place the plates in the freezer (-20°C) to cool them down to room temperature (10-15 min.). Use robot to add 280 µl cold 7.5M ammonium acetate. Cap and clamp tightly, invert and mix well (make certain that the pellets are not stuck to the bottom of the wells), let stand for 15 minutes at 4°C. Do not freeze.

6. Centrifuge samples (1 ml plates at 5,700 rpm, 6100g; 2ml plates at 4,000 rpm, 3,000g) for 15 min (30 min for 2ml plates) at 4°C to precipitate proteins and cell debris.

7. Measure distance from top of plate to the highest pellet. If distance is 28mm or greater, use the -26mm script, otherwise, use the -23mm script. Use robot to transfer the aqueous phase (450 µl) with a wide-bore pipette into labeled 1 ml isopropanol plates. Dry top of plate and cap with a newmat, clamp and invert plates 3 times. Place plates at 4°C for 30 min to overnight.

8. Centrifuge (1 ml plates at 5,700 rpm, 6100g) for 45 min at 4°C. Discard the supernatant by quickly and smoothly inverting the plate (over a pan to collect the isopropanol)and drying it on several layers of paper towel to remove excess liquid. Drip-dry inverted for a maximum of 1 min. Check that the pellets are not sliding out of plate.

9. First wash. Use robot to wash pellet by adding 500 µl 70% ethanol. Cap the plate withmats, roll mats on tightly, place plates in grinder and wet-grind at 30 cycles/sec for 10 sec. Do not remove top.

10. Centrifuge at 5,700 rpm for 15 min at 4°C. Discard ethanol by inverting plate(over a pan to collect the ethanol).

11. Second wash. Use robot to wash pellet by adding 500 µl 70% ethanol. Cap the plate withmats, roll mats on tightly, place plates in grinder and wet-grind at 30 cycles/sec for 10 sec.Do not remove top.

12. Centrifuge at 5,700 rpm for 15 min at 4°C. Discard ethanol by inverting plate(over a pan to collect the ethanol).

13. Drip-dry plate inverted on paper for a maximum of 1 min. DRY TOP OF PLATE, then centrifuge with NEW mats at 5,700 rpm for 1 min to settle pellets. Place plate in oven for 15-30 min or until all visible liquid is just barely gone. Do not over-dry in oven. Air-dry DNA pellet in hood at room temperature 3 h to ensure ethanol removal.

14. Use robot to re-suspend the DNA pellet in 208 µl autoclaved10mM Tris + TritonX-100, [Optional, but recommended: use Mantis to add 3 ul of RNase (10ug/ul) per sample]cap with a new top, place in 65°C water bath 30-60 min to denature remaining proteins. Vortex for 30 sec. Allow DNA to re-hydrate at 4°C overnight. Yield is approximately 20 ng/µl if diluted into 208 µl.

Extraction buffer (EB), 1 Liter, for 12 plates, 100mM Tris-HCl pH 8.0, 50mM EDTA pH 8.0, 1.25% SDS, 2mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 2% PolyVinylPolyPyrrolidone (PVPP). Do NOT Autoclave, make fresh each time.

i.~600 ml ddH2O

ii.0.573 gTCEP, do NOT exceed this amount, stir with heat until mixed before proceeding

iii.100 ml 1.0 M Tris-HCl pH 8.0

iv.100 ml 0.5M EDTA pH 8.0

v.12.5 gSodium Dodecyl Sulfate (SDS), stir until mixed before proceeding

vi.20 g PVPP (not soluble, must shake to keep mixed)

vii.Bring up to 1 Liter with ddH2O

Extraction buffer (EB), 500 ml, for 6 plates, 100mM Tris-HCl pH 8.0, 50mM EDTA pH 8.0, 1.25% SDS, 2mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 2% PolyVinylPolyPyrrolidone (PVPP). Do NOT Autoclave, make fresh each time.

i.~300 ml ddH2O

ii.0.2865 gTCEP, do NOT exceed this amount, stir with heat until mixed before proceeding

iii.50 ml 1.0 M Tris-HCl pH 8.0

iv.50 ml 0.5M EDTA pH 8.0

v.6.25gSodium Dodecyl Sulfate (SDS), stir until mixed before proceeding

vi.10 g PVPP (not soluble, must shake to keep mixed)

vii.Bring up to 500 ml with ddH2O

Ammonium Acetate (not a hazardous substance)

7.5M Ammonium Acetate (), 1 Liter, for 16 plates. Do NOT Autoclave, make fresh each time.

i.578.1 g Ammonium Acetate

ii.Bring up to 1 liter with ddH2O

7.5M Ammonium Acetate, 600 ml, for 8 plates. Do NOT Autoclave, make fresh each time.

i.346.86 g Ammonium Acetate

ii.Bring up to 600 ml with ddH2O

7.5M Ammonium Acetate, 500 ml, for 4 plates. Do NOT Autoclave, make fresh each time.

i.289.05 g Ammonium Acetate

ii.Bring up to 500 ml with ddH2O

70% Ethanol (ETOH), 1 Liter, Made from 95% ETOH, Store at RT

i.737 ml 95% Ethanol

ii.Bring up to 1 Liter with ddH2O

10mM Tris-HCl + 0.003125%TritonX-100, 1 Liter, from 1M Tris-HCl, pH 8.0, Autoclave, store at 4°C

i.10 ml 1.0 M Tris-HCl pH 8.0

ii.31.25 µl TritonX-100

iii.Bring up to 1 Liter with ddH2O

iv.Add stir-bar, stir vigorously until mixed

v.Autoclave, Store at 4°C


Suitable Plates for tissue collection and DNA extraction:

1ml 96-Deepwell plates

Best plates: Matrix plates use Matrix mats (Beckman mats fit very tightly, but are hard to remove).

Thermo Matrix, 1 ml, clear polypropylene, non-sterile, case of 40, Cat#4211-11

Mats:

Cat# 4410CS-MTX, Matrix Cap Mat 1ml - 100/case, $202.86/case, Need 3

MTX&v_xmp_i=35323

Cap Mats for 1ml 96 well plate, Nonsterile, Case of 12, #267002 (NOTE: Use these for the plates from Bioexpress, DO NOT USE BECKMAN PLATES, THEY LEAK!)

From VWR (

1ml Polypropylene Deep Well Microplates, Nonsterile, Case of 50, #40002-009

Mat Lids For 1.0 mL Microplates, Nonsterile, Case of 50,#40002-001

From Bioexpress (

1 ml Deep-Well Riplate, Polypropylene, Non-Sterile, 20/case, #P-8700-1. (NOTE: Use Beckman mats. Does NOT work with VWR mats.)

From Beckman (

Cap Mats for 1ml 96 well plate, Nonsterile, Case of 12, #267002 (NOTE: Use these for the plates from Bioexpress, DO NOT USE BECKMAN PLATES, THEY LEAK!)

From Bioexpress (

96 Deep-Well, 2.0 ml polypropylene plate, 50/pack, Cat#P-9641-2, $314.00

Silicone sealing Mat for 2.0 ml 96 Deep-Well Plate, 10/pk, T-3166-2, $60.00

P-8705-2, Racked Microtubes, Polypropylene, 1.1ml 8-strips, Natural, 10 Microracks of 96, $32.90

P-8705-1, 8-strip Microtubes, Polypropylene, 1.1ml 8-strips, Natural, 120 Tubestrips of 8, $19.90

P-8705-C, 8-strip Microtube Cap, Polyethylene, 120 CapStrips of 8, $9.70

P-8706-M, Microtube Mat, Polyethylene, 96-Well Plug Mats, 10 Microtube Mats, $20.90

For oven drying tissue, 35°C to 40°C, 2 to 4 days in oven, 50°C is TOO HOT.