DNA Extraction DNA Extraction (small scale) using CTAB method

Transcription

1 DNA Extraction DNA Extraction (small scale) using CTAB method This method is relatively simple, and has been used successfully with a wide range of monocot and dicot species. The method may be used with either fresh or dehydrated plant material. Although the DNA obtained is not of high purity, it is generally of sufficient quality for restriction analysis or for amplification by PCR. Yields are typically of the order of ug/g fresh weight of tissue. Protocol 1. Collect 1 g of leaf tissue and put in 1.5 ml tube. Homogenize the tissue using liquid nitrogen with a conical hand tissue grinder. Do not let the ground tissue to moist 2. Add 500 ul of 1.5x CTAB and mix well. Preheat 1.5x CTAB at 65 O C before using 3. Incubate for 1 hr. at 65 O C with occasional shaking. 4. Add 500 ul of chloroform:isoamylacol (24:1) and mix for 20 minutes on ice. 5. Centrifuge for 10 minutes at 10,000 rpm. 6. Transfer for upper phase into a news 1.5 ml tube. Add 50 ul of 10% CTAB and 1 volume chloroform:isoamylacol. Mix for 20 minutes on ice. 7. Centrifuge for 15 minutes at 10,000 rpm. 8. Transfer the upper phase to a new 1.5 ml tube. 9. Add 1 volume of CTAB precipitation buffer then mix by inverting. If DNA appears, hook out the DNA and transfer to a new 1.5 ml tube. If DNA does not appear, incubate at 65 O C for 30 minutes. As DNA precipitates, do not let it stand out 65 O C for more than 1 hour. Centrifuge at 12,000 rpm for 3 minutes and then discard the supernatant. 10. Add 300 ul of high salt TE and incubate at 65 O C until the pellet dissolves. 11. Add 2 volumes of 95% ETOH and mix gently. 12. Centrifuge at 14,000 rpm for 1 minute. 13. Wash the DNA twice with 70% ETOH by spinning at 14,000 rpm for 1 minute. 14. After the last wash discard the supernatant and centrifuge again at 14,000 rpm for 1 minute. Pipette out the excess ethanol. 15. Let ethanol and DNA to dry out then add TE buffer and store in 20 O C The amount of TE to be added depends on the amount of DNA Allow the DNA to dissolve in TE before storing in 20 O C 4

2 Reagents and solutions 4x CTAB (500 ml) for precipitation CTAB 20 g. 1 M Tris-Cl ph ml. 0.5 M EDTA ph ml. dh 2 O complete volume to 500 ml 3x CTAB (500 ml) for extraction buffer CTAB 15 g. 1 M Tris-Cl ph ml. 0.5 M EDTA ph ml. 5 M NaCl 210 ml. dh 2 O complete volume to 500 ml 2x CTAB Buffer 2% CTAB 1.4 M NaCl 1% PVP 0.1% Sodium meta bisulfite 20 mm EDTA 100 mm Tris ph % CTAB (200 ml) CTAB 20 g. 5 M NaCl 28 ml. dh 2 O complete volume to 200 ml High salt TE (1000 ml) 1 M Tris ph ml. 0.5 M EDTA ph ml. 5 M NaCl 200 ml. dh 2 O complete volume to 1000 ml -from 3x CTAB, dilute to prepare 1.5x CTAB -from 4x CTAB, dilute to prepare 1x CTAB 5

3 Extraction procedures for plant DNA must accomplish the following (Additional information to CTAB DNA extraction procedure). 1. The cell wall must be broken (or digested away) in order to release the cellular constituents. This is usually done by grinding the tissue in dry ice, liquid nitrogen or in hot buffer with a mortar and pestle or a food grinder. 2. The cell membranes must be disrupted, so that the DNA is released into the extraction buffer. This is accomplished by using a detergent, usually SDS (sodium dodecyl sulfate) or CTAB. 3. The DNA must be protected from the endogenous nucleases. The detergents are used for this purpose, as is EDTA (ethylenediaminetetraacetic acid). EDTA is a chelating agent that binds magnesium ions, generally considered a necessary cofactor for most nucleases. The DNA extracted from some tissues can be brown or gray in color. The purpose of the PVP included in the CTAB buffer in this method is to inhibit the polyphenol oxidase activity responsible for the coloration. 4. In addition, the buffer/tissue homogenate is emulsified with chloroform and/or phenol to denature and separate the proteins from the DNA. However all of the chloroform must be excluded from the collected aqueous fraction. 5. The DNA must be complexed with CTAB, present as CTAB-nucleic acid pellet and the CTAB will be removed from DNA by high salt TE. In the procedure, high salt concentration (1 M and above) are mandatory, since DNA/CTAB precipitates when salt concentrations are below M at temperatures below about 50 O C. 6. The DNA must be precipitated by 95% ethanol % ETOH will remove residual CTAB, salt and other contaminants. 8. Shearing of the DNA should be minimized. DNA in solution can be broken by exposure to turbulence. Typically, DNA Kb in length can be obtained without great care being taken. 9. The time between thawing of the frozen, pulverized tissue and its exposure to the extraction buffer should be minimized to avoid nucleolytic degradation of the DNA. A few commonly used procedures for the extraction of plant DNA are described. Two basic extraction protocols, the CTAB method and the Phenol/chloroform method, are presented which, in the part, have been used successfully with a wide range of species to give a good yield of reasonably high molecular weight DNA, albeit of variable purity. An alternative protocol is then presented for avoiding the use of dangerous chemicals, otherwise it s quick, simple and cheap. 6

4 Phenol/chloroform Extraction of DNA Phenol extraction is a common technique used to purify a DNA sample (1). Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol/chloroform interface and then is subjected to chloroform extraction to remove residual phenol. An equal volume of chloroform is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper layer is removed to new tube. The DNA is concentrated by ethanol precipitation. Protocol 1. Collect 1 g of leaf tissue and put in 1.5 ml tube. Pulverise the tissue under liquid nitrogen in a chilled pestle and motar. Add 500 ul of 1.5x CTAB buffer and mix well. 2. Add an equal volume of TE-saturated phenol:chloroform (1:1) to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for seconds. 3. Centrifuge the sample for 5 minutes at room temperature to separate the phases. 4. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol/chloroform interface. 5. (Steps 2-4 can be repeated until an interface is no longer visible) 6. To remove traces of phenol, add an equal volume of chloroform to the aqueous layer, and centrifuge for 3 minutes at room temperature. 7. Remove the upper aqueous layer to a new tube. 8. *Ethanol precipitate the DNA by adding volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. It is possible to place the sample at 20 O C overnight at this stage. 9. Centrifuge at 12,000 rpm in a microcentrifuge for 15 minutes at 4 O C, decant the supernatant, and drain inverted on a paper towel. 10. Add 80% ethanol (corresponding to about two volume of the original sample), incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above. 11. Place the tube in a Savant Speed-Vac and dry the DNA pellet for about 5-10 minutes, or until dry. 12. Always dissolve dried DNA in TE buffer. 13. It is advisable to aliquot the DNA purified in large-scale isolations (i.e. 100 ug or more) into several small (0.5 ml) microcentrifuge tubes for frozen storage because repeated freezing and thawing is not advisable. 7

5 * Typically, volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -2 0 C overnight (1). To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac. Reagents and solutions 3x CTAB (500 ml) for extraction buffer CTAB 15 g. 1 M Tris-Cl ph ml. 0.5 M EDTA ph ml. 5 M NaCl 210 ml. dh 2 O complete volume to 500 ml - 1.5x CTAB dilute from 3x CTAB TE buffer 10 mm Tris-HCl ph (25 O C) 0.1mM EDTA Phenol:Chloroform (1:1) Weigh out 20g phenol in a glass beaker. Add 20ml chloroform cover with cling film and mix well over a period of a few hours until all the phenol has dissolved. Store at 4 O C 8

6 DNA Extraction using DNA Trap This procedure can be quick, simple and cheap and avoid the use of dangerous chemicals. Protocol 1. Collect 1 g of leaf tissue and put in 1.5 ml tube. Homogenize the tissue using liquid nitrogen with a conical hand tissue grinder. Do not let the ground tissue to moist 2. Add 1,000 ul of extraction buffer and mix well. 3. Incubate for 1 hour at 65 O C 4. Put sample on ice for 5 minutes. 5. Add 100 ul of neutralizer and mix. 6. Put sample on ice for 5 minutes. 7. Centrifuge for 5 minutes at 14,000 rpm. 8. Transfer for upper phase into a new 1.5 ml tube. 9. Add 500 ul of trapping buffer and mix gently. 10. Centrifuge for 1 minute at 2,200 rpm. 11. Discard the supernatant solution. 12. Add 500 ul of washing buffer I and mix well. 13. Centrifuge for 1 minute at 2,200 rpm. 14. Discard the supernatant solution. 15. Add 500 ul of washing buffer II 16. Centrifuge for 30 sec. at 14,000 rpm. 17. Discard the supernatant solution. 18. Dry in an incubator for 1 hour at 65 O C or until all of the liquid has evaporated. 19. Re-hydrate the DNA in 100 ul of elution buffer and mix. 20. Incubate for 30 minutes at 65 O C 21. Centrifuge for 30 sec. at 14,000 rpm. 22. Transfer the supernatant solution into a new 0.2 ml tube. 9

7 Note: DNA trap protocol is owned by DNA Technology Laboratory. Please contact them for further information. 10

8 DNA Extraction using DNA Trap Collect 1 g of leaf tissue and put in 1.5 ml tube. Freeze the tissue using liquid nitrogen, grind it to find powder. Add 1,000 ul of Extraction buffer and mix well. Incubate for 1 hour at 65 o C. Put sample for 5 minutes on ice. Centrifuge for 5 minutes at 14,000 rpm. Add 100 ul of neutralizing buffer and mix well. Put sample for 5 minutes on ice. Transfer for upper phase into a new 1.5 ml tube. Add 500 ul of trapping buffer and mix gently. 11

9 Centrifuge for 1 minutes at 2,200 rpm. Discard the supernatant solution. Add 500 ul of washing buffer I and mix well. Centrifuge for 1 minutes at 2,200 rpm. Discard the supernatant solution. Add 500 ul of washing buffer II Centrifuge for 30 sec. at 14,000 rpm. Discard the supernatant solution. 12

10 Dry in an incubator for 1 hour at 65 o C or until all of the liquid has evaporated. Rehydrate the DNA in 100 ul of elution buffer and mix well. Incubate for 30 minutes at 65 o C. Centrifuge for 30 sec. at 14,000 rpm. Transfer the supernatant solution into a new 0.2 ml tube. 13

11 DNA Extraction from rice grain 1. Add 200 ul of extraction buffer in microtube containing 1 grain. Incubate for 1 hour at 65 O C. 2. Grind the sample then add 500 ul of extraction buffer and incubate for 30 min at 65 O C. 3. Add 100 ul neutralizer and gently mix then keep on ice for 10 min. 4. Centrifuge at 14,000 rpm for 10 min. 5. Take 500 ul of supernatant into new microtube, then add 500 ul of trapping buffer solution. Shake for 10 min then spin down for 15 sec. 6. Remove supernatant then clean with 500 ul washing buffer I for 2 times. Mix by vortexing then spin down and remove supernatant and repeat washing. 7. Clean the precipitate with 500 ul washing buffer II then dry at 65 O C for 30 min. Add 100 ul elution buffer and incubate for 30 min at 65 O C, centrifuge at 14,000 rpm for 5 min. Take the supernatant to use. Note: DNA trap protocol was formulated by DNA Technology Laboratory, thus additional information regarding the protocol can be obtained from the said laboratory. References S. Wilkie (1996) Genomic DNA Isolation, Southern Blotting and Hybridization. In: Melody S. Clark (ed)plant Molecular Biology A Laboratory Manual, Springer, New York, pp 3-9 Scott O. Rogers and Arnold J. Bendich (1994) Extraction of total cellular DNA from plant, algae and fungi. Plant Molecular Biology Manual D1: 1-8 Simon A. J. Warner (1996) Genomic DNA Isolation and Lambda Library Construction. In: Gary D. Foster and David Twell (ed) Plant Gene Isolation, John Wiley & Sons, England, pp