Harmer Lab:Protocols:Direct PCR

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Room 2123
Department of Plant Biology
1002 Life Sciences, One Shields Ave.
University of California Davis
Davis, CA 95616

Contact: slharmer at ucdavis.edu

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96-Well Format DNA Extraction for Direct PCR

Modified from Bellstedt et al. 2010 by Matt

  1. Add a single ball bearing to each 1.2 ml tube containing tissue (1 cotyledon sized piece works, up to 1 small leaf normally)
  2. Add 350μL grinding buffer
  3. Disrupt tissue by shaking with paint shaker for 1 min. Repeat if clumps of tissue remain
  4. Spin for 5min @ 3000rpm
  5. Add 4μL of extract to 25μL GES buffer in PCR tube
  6. 95°C for 10 min, 4°C for 5 min
  7. Add 0.5μL of final extract to standard PCR reaction (20μL)

100mL Grinding Buffer

To prepare 100 mL of grinding buffer dissolve all components except Tween 20 in double-distilled sterile water. Stir at low speed to avoid excessive foaming. Once all components have dissolved, adjust pH to 9.6 using NaOH solution. Then add Tween 20. Autoclave and store at 4°C.

  1. Na2CO3 (Sodium Carbonate) 0.159g
  2. NaHCO3 (Sodium Hydrogen Carbonate) 0.293g
  3. PVP 40 (Polyvinylpyrrolidone) 2g
  4. BSA (Bovine Serum Albumin) 0.2g
  5. Tween 20 50μL
  6. Na2S2O5 (Sodium Metabisulphite) 1g

100mL GES Buffer

  1. 1M Glycine, pH9, 10mL
  2. 2M NaCl, 2.5mL
  3. 0.5M EDTA, pH8 200μL
  4. 500μL Triton X-100