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Transcript
Sunflower DNA Extraction (CTAB) by Dr. Shunxue Tang in SJK’s Lab DNA Extraction for Sunflower PCR and RFLP Analysis (Small Quantity) 1. Weigh out 50-100 mg lyophilize tissue powder to 2.0 mL tube, and add 800 uL of 1x CTAB extraction buffer (1% CTAB, 0.7 M NaCl, 50 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 0.5% PVP40, autoclaved and store at RT) preheated at 60°C and 1 uL βMercaptoethanol (0.1%-0.3%). 2. Put the 2.0 mL tube in 60-65°C water bath for 1.5 hr, mix gently by inverting the tubes for several times every 20 min. Take out the tube and cool to RT 3. Add an equal volume (around 800 uL) of chloroform: isoamyl alcohol (24:1 V/V) and mix by slight inversion to form an emulsion. Centrifuge by 10,000 rpm for 15 min at RT. Carefully transfer the top aqueous solution to a new 2.0 mL centrifuge tube 4. Add an EQUAL volume (around 700 uL) of 1x CTAB precipitation buffer (1% CTAB, 50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, autoclaved and store at RT) to the top aqueous phase and mix it by inverting several times. The DNA-CTAB compounds will be precipitated in 1-5 min at RT. Put the tubes in 4°C for several hours if precipitates were not observed at RT 5. Centrifuge at 5,000 rpm for 5 min, pour off supernatant. Add 400 uL 1M NaCl to the pellet; incubate in a 50°C water bath till the pellet dissolved. Slowly pipette the pellet up and down for several times to speed the process 6. Add 2 uL DNase-free RNase A (10 mg/mL) to the dissolved DNA solution. Incubate at 50°C water bath for 1 hr 7. Transfer the above DNA solution to 2 volumes of pre-cooled (-20°C) 100% ethanol in 2.0 mL tube with a wide-bore pipette tip; invert several times to mix the solution. Precipitate the DNA for 30 min at -20°C 8. Hook out the DNA using tips, and briefly wash the DNA one time with 70% ethanol. Then, wash the DNA with 70% ethanol for at least 2 hr at RT (dissolve the residual CTAB) 9. Hook out the DNA and dry the DNA pellet in an air-hood at RT. Dissolve the DNA in 200 uL ddH2O or 1x TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0, autoclaved); put the DNA in 50°C incubator for 1 or 2 hrs will speed this process. Measure the DNA concentration, which should be around 0.5-1 μg/μL. Check the DNA quality on 1.0% agarose gel. An intact band should be observed around 50-80 kb (no RNA will be observed) Notes 1. Sunflower is a recalcitrant plant to obtain qualified DNA for RFLP analysis because of its high polyphenols, polysaccharides and tannins. SDS-KAc method and common CTAB method (precipitate DNA directly from DNA-CTAB buffer using isopropanol) can not produce highly purified DNA because DNA co-precipitates with above secondary metabolites. This modified CTAB method is relatively simple, and can provide high quality and quantity DNA for RFLP analysis. Usually more than 200 ug DNA can be obtained from 1 g fresh young leaf, and the DNA size is around 50 kb 2. CTAB-DNA complexes are soluble in high salt (0.7 M NaCl), and can be precipitated by lowering the salt to 0.35 M NaCl. Polyphenols, residual proteins and many polysaccharides remain in the supernatant. The soluble PVP 40 can remove the polyphenols, and high NaCl can remove polysaccharides 3. All items used for DNA isolation should be autoclaved 4. Never shake the DNA solution violently to avoid mechanical shear of DNA 1 Sunflower DNA Extraction (CTAB) by Dr. Shunxue Tang in SJK’s Lab 5. DNA purity is very important for RFLP analysis. The DNA solution may turn yellow or the DNA is hard to dissolve after storage if too much polysaccharide and polyphenol co-precipitate with DNA. These materials will inhibit the DNA digestion (resulting in partial digestion), which results in extra hybridization bands in Southern Blotting Solutions 1. Extraction Buffer (EB) autoclaved for lyophilized tissue (1x CTAB) 100 mL 5.0 14.0 4.0 1 0.5 0.1 500 mL 25 70 20 5 2.5 0.5 1000 mL 50 140 40 10 5.0 1.0 50 mL 5.0 14 2.0 1g 0.5 0.1 100 mL 10 28 4.0 2g 1.0 0.2 500 mL 50 140 20 10 g 5.0 1.0 1000 mL 100 280 40 20g 10.0 2.0 Precipitation Buffer (PB) autoclaved and store at RT (1x CTAB) Stock 50 mL 50 Mm Tris-HCl pH 8.0 (mL) 1M 2.5 10 mM EDTA (mL) 0.5 M 1.0 1% (w/v) CTAB (g) Powder 0.5 100mL 5.0 2.0 1 500 mL 25 10 5 1000 mL 50 20 10 50 mM Tris-HCl pH 8.0 (mL) 700 mM NaCl (mL) 20 mM EDTA (mL) 1% (w/v) CTAB (Sigma, g) 0.5% PVP40 (Sigma, g) 0.1% BME 1uL/mL (add just before use) Stock 1M 5M 0.5 M Powder Powder Liquid 50 mL 2.5 7.0 2.0 0.5 0.25 0.05 Extraction Buffer (EB) autoclaved for fresh tissue (2x CTAB) 100 mM Tris-HCl pH 8.0 (mL) 1.4 M NaCl (mL) 20 mM EDTA (mL) 2% (w/v) CTAB (Sigma, g) 1.0% PVP40 (Sigma, g) 0.2% BME 2uL/mL (add just before use) Stock 1M 5M 0.5 M Powder Powder Liquid 2. 2