(08-07-2017, 04:05 AM)nasir Wrote: hi all my friends i kindly request you to help me with CTAP procedure to an isolation of Genomic DNA from Marine sample,
Thanks in advance
DNA EXTRACTION FROM MARINE ALGAE AND SEAGRASSES
There are several published extraction methods that work well with marine algae and which have influenced the method described below (Coyer et al. 1994; Doyle and Doyle, 1987; Fain et al. 1988; Goff and Coleman, 1988; Herrin and Worley, 1990; Murray and Thompson, 1980; Polne-Fuller, 1991). This extraction protocol has yielded large quantities of DNA from a variety of algal species. For some species, the DNA can be used following ethanol precipitation but others require further purification of CsCl gradients for reproducibly high quality DNA.
DNA extraction from marine algae and seagrass is hampered by the large quantity of polysaccharides and polyphenolics produced within the thalli (leaves) of many species. The method uses the detergent CTAB and high salt concentrations to precipitate polysaccharides. CTAB forms complexes with polysaccharides at salt concentrations above 0.5 M at room temperature (Murray and Thompson, 1980). At lower salt concentrations, CTAB complexes with nucleic acids. PVPP and beta-mercaptoethanol are added to bind and precipitate polyphenolics.
Many steps in this protocol should be optimized for different algae depending on the characteristics of the thallus and polyphenolic content, but hopefully this outline will provide a starting point. The protocol assumes that the users are familiar with the general molecular procedures including the handling and disposal of chemicals.
2x CTAB Buffer
100 mM Tris pH 8.0
1.4 M NaCl
20 mM EDTA
Add beta-mercaptoethanol to 0.2% (v/v) daily.
10 mM Tris (pH 8.0)
0.1 mM EDTA
Solutions to have on hand:
5 M NaCl
7.5 M Ammonium acetate
3.0 M Sodium acetate pH 5.2
TE saturated butanol
10 mg/mL ethidium bromide (suspected carcinogen)
Ethanol (absolute and 70%)
25:24:1 phenol:chloroform:isoamyl alchohol
24:1 chloroform:isoamyl alcohol
Mortar and pestle
50 mL polypropylene screw cap tubes
30 mL and 15 mL tubes
1.5 mL centrifuge tubes
Sterile pasteur pipets
Rinse the thalli in sterile seawater and then blot dry. If needed gently clean the surface of the tissue with a razor blade. Chop finely, freeze in liquid nitrogen. Grind approximately 1 gm of tissue in liquid nitrogen cooled motar and pestle.Transfer to 50 mL screwcap tube and add 8 mL of 2x CTAB. Mix tissue well and then add 10% SDS to a final concentration of 0.1%.Incubate at 60 C for 0.5-1 h. Periodically, mix gently during the incubation.The time of incubation should be optimized for each species. We have also found that room temperature incubation results in perhaps slightly lower yields of DNA but significantly lower polysaccharide contamination. Treat the lysates gently to reduce shear forces.Add an equal volume of chloroform:isoamyl alcohol (24:1), mix very well.Centrifuge in a clinical centrifuge at top speed ( ca. 2,000 x g) for 10 min. The interface should be solid at the end of the spin, if not, increase spin time. The DNA remains in the upper (aqueous layer).Repeat chloroform:isoamyl alcohol extraction until the interface is clean.Add 2/3 volumes of isopropanol, mix gently.Centrifuge for 15 min at 10,000 x g. Discard supernatant and wash pellet with 70% ethanol.Air dry pellet. Resuspend in TE to appropriate concentration. DNA ma be shaken gently at room temperature or at 65 C for several hours to facilitate solubilization.If not further purifying DNA on CsCl gradient, remove RNA by adding 0.1% SDS and 1 µg/mL DNAse-free RNAse (final concentrations) and incubating for 0.5-1 h at 37 C. Extract once with an equal volume of phenol:chloroform:isoamyl alcohol . Centrifuge 10 min at 10,000 x g. Transfer to clean tube and precipitate with ethanol as described in the next step.DNA can be further purified by precipitation with 1/3 volume of 7.5 M ammonium acetate and 2.5 volumes of ethanol.Centrifuge as above, wash with 70% ethanol, and air dry. Resuspend in TE.
This protocol is modified from that published by Herrin and Worley (1990) and Fain et al. (1988) and is designed for the TL-100 table-top ultracentrifuge. The technique can however, be adapted to any ultracentrifuge.
Adjust the volume of the DNA sample to 2.0 mL TE. Add 1 gm/mL CsCl. Mix well and allow the solution to return to room temperature.Transfer to 3 mL polyallomar ultracentrifuge tubes and spin at 25,000 x g for 25 minutes at 20 C. At this there may be a visible pellet of polysaccharides, proteins and RNA. The DNA is still in solution. If the alga you are working with does not have many of polysaccharide material, this step can be omitted.Add ethidium bromide to a final concentration of 100 ug/mL to the quick seal ultracentrifuge tubes. Then add the CsCl supernatant from above using a sterile Pasteur pipet. Seal the tubes following the manufacturer instructions.Centrifuge for 4 h at 100,000 rpm.Visualize the bands with long wave UV light. Remove the bands using an 18 gauge needle attached to a 1 mL syringe.Extract the DNA with an equal volume of butanol (saturated with TE) repeatedly until all of the dye is removed.Add two volumes of TE and NaCl to 0.2 M and mix thoroughly (salt can be omitted with pellets seem overly salty after precipitation).Add 2.5 volumes ethanol and store overnight at -20 C.Centrifuge for 30 min at 10,000 xg . Wash with 70% ethanol and air dry.Resuspend in 50 to 100 uL of TE. DNA can be reprecipitated as described in the last step of the previous section.