DNA purification from saliva
Материал из Zbio
DNA extraction from buccal cells using Gentra Puregene Tissue Kit
- Check that you have:
- >> Gentra Puregene Tissue Kit QIAGEN (cat#158622)
- >> 100% Isopropanol
- >> 50mL polypropylene tube per sample
- >> 1.5 mL tube per sample
- >> centrifuge, for example eppendorf 5810 R
- >> water bath
- >> rotator
- Collect buccal cells
Prepare 20mL TS per sample.
Collect the buccal cells by swishing orally the mouthwash 20 times and spitting back into the 50-mL tube.
- Lyse cells
Centrifuge at 2000 g for 10 min and pour off the supernatant. Leave behind about 100µl of residual liquid.
Vortex vigorously for 5 seconds to resuspend the cells in the residual supernatant.
Add 3 mL of cell lysis solution and vortex 5 sec at medium speed.
Add 15 µL of proteinase K and incubate the lysate at 55°C for 1 h.
- RNase A treatment
Add 15 mL of RNase A solution to the cell lysate.
Mix the sample by inverting the tube 25 times and incubate at 37°C for 15 min.
- Protein precipitation
Cool the sample to RT.
Add 1 mL of protein precipitation solution.
Vortex at high speed for 20 sec and place the tube in an ice bath for 10 min.
Centrifuge at 2000 g for 10 min.
- DNA precipitation
Pour the supernatant with DNA to a clean 50-mL tube containing 3 ml of 100% isopropanol and 5 µL glycogen solution.
Mix the sample by inverting it gently 50 times and keep the tube at RT for 5 min.
Centrifuge at 2000 g for 10 min.
Pour off the supernatant and drain the tube.
Add 3 mL of 70% ethanol and invert the tube several times to wash the DNA pellet.
Centrifuge at 2000 g for 3 min and pour off the ethanol.
Invert and drain the tube and allow to air dry for 15 min.
- DNA hydration
Add 200 µL of DNA hydration solution.
Allow DNA to rehydrate by incubating at 65°C for 1 h and/or overnight at RT. Tap the tube periodically or place it on a rotator to aid in dispersing the DNA.
Vortex the sample, centrifuge brieﬂy, and transfer it to a 1.5-mL tube.
Store DNA at 2°C to 8°C. For long-term storage, store at 20°C or 80°C
- Solutions (*add calculation tables, Lesha)
1. Contents of the Gentra Puregene Tissue Kit:
- Cell lysis solution
- Proteinase K (20 mg/mL).
- Protein precipitation solution
- DNA hydration solution
- RNase A solution
2. Transport solution (TS) (100mM NaCl, 10mM TrisCl, pH 8.0).
3. 70% Ethanol
4. Glycogen (20 mg/mL)
- ↑ Gentra Puregene Tissue Kit, modifications to the Kit protocol are according to the article (Arch Pathol Lab Med. 2001;125:127–133). Generally, Kit is able to give sufficient yield but combining it with the article protocol improves significantly the amount of DNA to be obtained. Average DNA yield from mouthwash is about 50 µg that was checked by our lab several times.
- ↑ For best results for collecting Buccal cells:
Evening before: brush thoroughly teeth and tongue, so that no food rests come into the mousewash;
In the morning before brushing teeth, eating or drinking:
- rub the inner sides of the cheeks with a clean spatula or spoon
- swish the TS from 50 mL tube vigorously in the mouth for 20–30 sec, then spit it back into the same tube.
Buccal cells are stable at RT for at least 7 days in the mouthwash solution.
- ↑ Loose pellets will result if the samples sit too long after centrifugation
- ↑ Thorough cell resuspension in the residual supernatant will greatly facilitate cell lysis
- ↑ Vortexing on this step helps to maximize contact between the cells and the cell lysis solution
- ↑ Ensure that you have a tight pellet
- ↑ Glycogen solution (20 mg/mL)
- ↑ The DNA may or may not be visible as a small white pellet, depending on yield
- ↑ 200 µL will give a concentration of 100 ng/µL if the total yield is 20 µg of DNA