I need to isolate pure DNA without using kit, please tell me procedure if anyone knows?
In brief,
Tell donor to keep oral cavity 30 minutes stable, no eating drinking for 30 minutes, Then collect drop by drop saliva by directly attaching collective tube a the tip of the lower lip, you can get pure saliva.
This you can use for protein as well as for DNA analysis. If you use kits, then its easy to do otherwise, If kits are not available, I advise DNAzol method, which is best universal method for DNA extraction from any source. For DNA isolation, just use same volume 1:1 (250 ul Saliva : 250 ul DNAzol) by absolute ethanol precipitation and 70% ethanol washing, air drying solubilize in TE buffer, Add RNase incubate, deactivate DNase activity for keeping at 60 degree for 20 minutes and store in freezing conditions or use directly for PCR.
HI, You can use DNA extraction kits commercially available. Those are easy-going.
Best, A.
In brief,
Tell donor to keep oral cavity 30 minutes stable, no eating drinking for 30 minutes, Then collect drop by drop saliva by directly attaching collective tube a the tip of the lower lip, you can get pure saliva.
This you can use for protein as well as for DNA analysis. If you use kits, then its easy to do otherwise, If kits are not available, I advise DNAzol method, which is best universal method for DNA extraction from any source. For DNA isolation, just use same volume 1:1 (250 ul Saliva : 250 ul DNAzol) by absolute ethanol precipitation and 70% ethanol washing, air drying solubilize in TE buffer, Add RNase incubate, deactivate DNase activity for keeping at 60 degree for 20 minutes and store in freezing conditions or use directly for PCR.
In brief, you can use salting-out procedure. The key is use 50ul of saliva and add 250ul of lysis buffer (50mM Tris-Cl, 10mM EDTA, pH8.0; 2% SDS). Add 1.5ul of 20mg/ml Proteinase-K. Incubate at 65C for 30 minutes. Tap tube occasionally.
Cool sample to room temperature. Add 5M NaCl, vortex at high speed. Incubate on ice for 8 minutes. Centrifuge at high speed for 5 minutes. A pellet should form.
Transfer 300ul of the supernatant to a new tube containing 300ul of Isopropanol. Invert tube gently for 50 times. Centrifuge at high speed for 5 minutes. Discard supernatant carefully without dislodging the pellet.
Add 300ul of 70% EtOH and centrifuge at high speed for 5 minutes. Discard supernatant carefully without dislodging the pellet. Air-dry tube for 15 minutes, add 50ul TE. Rehydrate at 55C for 15 minutes.
DNA extraction from saliva
First, we need to collect five milliliters of saliva. The samples must be divided into one 2 ml and three 1 ml aliquots. Each aliquot needs to be mixed with an equal volume of lysis buffer (50 mM Tris, pH 8.0, 50 mM EDTA, 50 mM sucrose, 100 mM NaCl, 1% SDS). DNA extraction thirty microliters of proteinase K (20 mg/ml) and 150 µl of 10% SDS are added to 2 ml of saliva extraction buffer mixture that was previously incubated overnight in a shaking water bath at 53°C. Later 400 µl of a solution 5M NaCl need to be added and the solution needs to be incubated for 10 min on ice. Subsequently the mixture must be distributed equally into 2 ml centrifuge tubes and centrifuged for 10 min at 13,000 rpm. The supernatant needs to be transferred to a new tube to which 800 µl of isopropanol must be added, later the tubes need to be incubated 10 min at room temperature and centrifuged for 15 min at 13,000 rpm. The supernatant must be discarded and the pellets need to be washed once with 500 µl of 70% ethanol, then the pellets must be dried and dissolved in 30 µl of double distilled water. Keep on PCR protocol.
Reference: Quinque, D., Kittler, R., Kayser, M., Stoneking, I., & Nasidze, I. ( 2006). Evaluation of saliva as a source of human DNA for population and association studies. Analytical Biochemistry . Quinque, D., Kittler, R., Kayser, M., Stoneking, I., & Nasidze, I. (Marzo de 2006). Evaluation of saliva as a source of human DNA for population and association studies. Analytical Biochemistry .
Dear colleagues, could you please give any advice on a buffer for RT storage of saliva for further DNA extraction? I suppose, i need to add antimicrobials, but NaAz, for example, is toxic - maybe, some other suggestions? What about -20C freezing - is it enough just to collect the samples, put them to -20 and then several days ago just perform ph-cl extraction? Or i'd better add some salts to stabilize?
You can directly transfer the saliva into the lysis buffer. But without Proteinase-K. Can store at room temp.
Ag Muhammad Sagaf Abu Bakar, thank you very much - so SDS in the buffer will be enough for bacterial lysis, i suppose? But what about enzymes?
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