Download Sunflower DNA extraction for RFLP and PCR

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