E.Z.N.A.?ProtocolforMouseTailsSnips

實驗概要

The E.Z.N.A.^? Tissue DNA Kit provides a rapid and easy method for the isolation of genomic DNA for consistent PCR and Southern analysis. Up to 30 mg tissue or up to 1 cm sections of mouse tail can be readily processed in one time. The method can also be used for preparation of genomic DNA from mouse tail snips, blood, buffy coat, serum, and plasma. The kit allows single or multiple, simultaneous processing of samples. There is no need for phenol/chloroform extractions, and timeconsuming steps such as precipitation with isopropanol or ethanol, are eliminated. DNA purified using the E.Z.N.A.^? Tissue DNA method is ready for applications such as PCR*, Southern blotting, and restriction digestion.

主要試劑

1. Elution Buffer (~0,5 ml per sample) equilibrated to 70°C.

2. For each sample, premix 200 ul Buffer BL with 200 ul absolute ethanol and vortex. This can be prepared fresh, or pre-made and stored at room temperature. Do not store this mixture for more than 1 month.

3. Absolute ethanol - approximately 0.3 ml per sample.

4. RNase A (Optional) - stock solution at 25 mg/ml.

主要設備

1. Tabletop microcentrifuge and sterile 1.5 ml tubes.

2. Shaking waterbath set to 55°C.

3. Have a shaking waterbath set to 55°C.

實驗步驟

Bring frozen samples and OB Protease solution to room temperature and, preheat an aliquot of Elution Buffer (approximately 0.5 ml per sample) at 70°C.

1. Snip two pieces of mouse tail 0.2 - 0.5 cm in length, place into a sterile 1.5 ml microcentrifuge tube, and add 180 ul of Buffer TL. If necessary cauterize the wound to stop bleeding. Having appropriately earmarked the animal, return it to a clean cage.

Note: Mice should not be older that 6 weeks, since lysis will be more difficult resulting in suboptimal DNA yields. If possible, obtain tail biopsy at 2-4 weeks and freeze samples at -70°C until DNA is extracted.

Add 25 ul of OB protease and vortex to mix. Incubate in a 55°C shaking waterbath for 1-4 hours or until lysis is complete. If no shaking waterbath is available, vortex vigorously every 20-30 min. Incomplete lysis may block column flow and significantly reduce DNA yields. Incubation time for complete tail lysis is dependent on length of tail and age of animal; 0.5 cm tail pieces from 2 week-old mice typically lyse in approximately 2 hours. For older animals an overnight incubation may improve yields. Note that bone and hair will not lyse.

2. Centrifuge for 5 min at 10,000 x g to pellet insoluble tissue debris and hair. Carefully aspirate the supernatant and transfer to a sterile microfuge tube leaving behind any insoluble pellet.

OPTIONAL: Mouse tail tissue contains RNA that can co-purify with the DNA. This will not interfere with PCR reactions, but other enzymatic reactions may be affected. To remove RNA, add 15 ul of RNase A (25 mg/ml) and incubate 2 min at room temperature.

3. Add ONE volune of BL followed by ONE volume of ethanol. Alternatively, user can add 2 volume of premixed BL/ethanol mixture to the sample. Vortex thoroughly to mix at maxi speed for 15 sec. Thoroughly mixing is essential at this point. If precipitation can be seen at this point, break the precipitation by pipetting up and down 10 times.

4. Assemble a HiBind?DNA column in a 2 ml collection tube (provided). Transfer the entire lysate from step 6 into the column including any precipitate that may have formed. Centrifuge at 8,000 x g for 1 min to bind DNA. Discard flow-through liquid.

5. (Optional) If greater than 30 mg tissue is used, repeat transfer the remaining lysate into the column and centrifuge as above. Make sure that all of the lysate has pass through the column.

6. Place the column into a second 2 ml collection tube and wash by pipetting 500 ul of Buffer HB. Centrifuge at 8,000 x g for 1 min. Discard flow-through liquid and 2ml collection tube.

7. Place the column into a second 2 ml collection tube and wash by pipetting 700 ul of DNA Wash Buffer diluted with ethanol. Centrifuge at 8,000 x g for 1 min. Discard flow-through liquid and re-use 2ml collection tube in next step.

Note that DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle or page 3. If refrigerated, the diluted DNA wash buffer must be brought to room temperature before use.

8. Place the column back into the 2ml collection tube from step 10, wash the column with a second 700 ul of DNA Wash Buffer diluted with ethanol and centrifuge as above. Discard flow-through.

9. Place the column back into the same 2 ml collection tube, centrifuge the empty column at maximum speed (>12,000 x g) for 2 min to dry the column. This step is crucial for ensuring optimal elution in the following step.

10. Place the column into a sterile 1.5 ml microfuge tube and add 50-200 ul of preheated (70°C) Elution Buffer. Allow tubes to sit for 3 min at room temperature.

11. To elute DNA from the column, centrifuge at 10,000 x g for 1 min. Repeat the elution with a second 100-200 ul of Elution Buffer.

Note: Each 100-200 ul elution typically yields 60-70% of the DNA bound to the column. Thus two elutions generally give ~90%. However, increasing elution volume reduces the concentration of the final product. To obtain DNA at higher concentrations, elution can be carried out using 50 ul to 100 ul Elution Buffer (which slightly reduces overall DNA yield). Volumes lower than 50 ul greatly reduce yields. In some instances yields may be increased by incubating the column at 70°C (rather than at room temperature) upon addition of Elution Buffer. If necessary the DNA can be concentrated. Add sodium chloride to a final concentration of 0.1 M followed by 2X volume of absolute (100%) ethanol. Mix well and incubate at -20°C for 10 min. Centrifuge at 10,000 x g for 15 min and discard supernatant. Add 700 ul of 80% ethanol and centrifuge at 10,000 x g for 2 min. Discard supernatant, air dry the pellet (2 min) and resuspend DNA in 20 ul sterile deionized water or 10 mM Tris-HCl, pH 8.0.