E.Z.N.A.® Protocol for Isolation of Genomic DNA from Swab
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实验原理
The E.Z.N.A.® MicroElute Genomic DNA Kit uses the reversible binding properties of the HiBind® matrix, a new silica-based material, combined with the MicroElute column spin technology which allows smaller elution volume as little as 10ul. A specially formulated buffer system allows genomic DNA up to 40 kb to bind to the matrix. Samples are first lysed under denaturing conditions and then applied to the HiBind® Mircro-spin columns to bind DNA, while cellular debris, hemoglobin, and other proteins are effectively washed away. High quality DNA is finally eluted in sterile deionized water or low salt buffer.
实验试剂
2. DTT (for processing hair and semen)
3. Elution Buffer or ddH2 O pre-warmed at 70°C
实验设备
1. 1.5 ml or 2ml microcentrifuge tubes
2. Water Bath or heating block preset at 60°C
3. Water Bath or heating block preset at 70°C
4. Microcentrifuge with rotor for 2ml tubes
5. Tabletop centrifuge capable of 20,000 x g (13,000 x rpm)
6. Optional: Omega Homogenizer Column (HCR-01) for collect any remaining liquid from paper or swab.
实验步骤
1. Place the swab in a 2 ml microcentrifuge tube. 2. Add 600ul Buffer TL and 20ul Protease solution into the tube. Mix throughly by vortexing for 30 seconds at maxi speed.
3. Incubate the tube in a heating block or a waterbath at 55°C for at least 1 hour. Mix the sample few times during the incubation by briefly vortexing.
4. Briefly centrifuge the tube to spin down any liquid drop from inside of the lid.
5. Add 620ul Buffer BL, close the lid, mix throughly by vortexing. If Linear Acrylamide is needed, add 4ul of dissolved Linear Acrylamide to 660ul Buffer BL. See page 3 for detailed instruction.
6. Place the tube in a heating block or waterbath preset at 70°C. Incubate for 10 minutes. Vortex the tube 10 seconds few times during incubation.
7. Briefly centrifuge the centrifuge tube to bring down any liquid drop from inside of the lid.
8. Add 620 ul absolute ethanol and mix thoroughly by vortexing for 20s at maxi speed. Centrifuge briefly to bring down any liquid from inside of lid.
9. Assemble a HiBind® MicroElute column in a 2 ml collection tube (provided). Transfer the 700ul of lysate from Step 8 into the column including any precipitate that may have formed. Close the lid and centrifuge at 8,000 x g for 1 min to bind DNA. Discard flow-through liquid and re-use the collection tube.
10. Place the HiBind® MicroElute column into the same collection tube from step 9 and repeat step 9 until all of the remaining lysate from step 8 has passed through the HiBind® MicroElute column. Discard the flow-through and collection tube.
Note: For maximum yield, Collection any remaining liquid from swab, transfer all swab into a Homogenizer Column (not supplied) and centrifuge at 20,000 x g for 2 minutes to collect remaining lysates. Homogenizer column can be purchased separately from Omega Bio-tek (Product No. HCR-001 an HCR-003).
11. Place the column into a new collection tube (supplied). Add 500ul of Buffer HB in the column. Close the lid and centrifuge at 8000 x g for 1 minute. Discard the flow-through and collection tube.
12. Place the column into a new 2 ml tube (supplied) and wash by pipetting 650 ul of DNA Wash Buffer diluted with ethanol . Centrifuge at 8,000 x g for 1 min. Discard flow-through liquid and re-use the collection tube.
Note: DNA Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions or on Page 3 for preparation.
13. Using the same collection tube from step 12, wash the column with a second 650 ul of DNA Wash Buffer diluted with ethanol and centrifuge as above. Discard flow-through and re-use the collection tube.
14. Using the same 2ml collection tube, centrifuge empty column at maximum speed (20,000 x g) for 3 min to dry the HiBind® membrane. This step is crucial for ensuring optimal elution in the following step.
15. Place the column into a sterile 1.5 ml microfuge tube and add 10-50ul of preheated (70°C) Elution Buffer or water onto the center of the membrane. Allow tubes to sit for 3 min at room temperature.
16. To elute DNA from the column, centrifuge at 20,000 x g for 1 min.
