Harmer Lab:Protocols:Direct PCR

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 Add a single ball bearing to each 1.2 ml tube containing tissue (1 cotyledon sized piece works, up to 1 small leaf normally) Add 350μL grinding buffer Disrupt tissue by shaking with paint shaker for 1 min. Repeat if clumps of tissue remain Spin for 5min @ 3000rpm Add 4μL of extract to 25μL GES buffer in PCR tube 95°C for 10 min, 4°C for 5 min 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. Na2CO3 (Sodium Carbonate) 0.159g NaHCO3 (Sodium Hydrogen Carbonate) 0.293g PVP 40 (Polyvinylpyrrolidone) 2g BSA (Bovine Serum Albumin) 0.2g Tween 20 50μL Na2S2O5 (Sodium Metabisulphite) 1g 100mL GES Buffer 1M Glycine, pH9, 10mL 2M NaCl, 2.5mL 0.5M EDTA, pH8 200μL 500μL Triton X-100