Extraction of DNA from tissue: High salt method.

Transcription

1 Extraction of DNA from tissue: High salt method. Version 1.0, September Animal Genomics Laboratory, Lab 1.03g, Donnan Laboratories, Crown Street, School of Biological Sciences, University of Liverpool, L69 7ZD. UK. This protocol was developed from that provided at: Any comments or suggestions should be sent to Phill Watts at

2 Materials Required: TNES buffer, Proteinase-K, 100 % ethanol, 70 % ethanol, sterile distilled water, Tris-EDTA, 1

3 1. Turn on a water bath to 55ºC. 2. Take a small amount of tissue (~ 0.5 cm 2 ) and chop it (as finely as you can be bothered) with a sterile scalpel blade. Transfer the sample to a labeled 1.5 ml microcentrifuge tube. [Using too much tissue can give poorer quality DNA and a worse yield]. 3. Add 600 µl of TNES buffer and 35 µl of Proteinase-K (20 mg/ml). Mix the sample by inverting the tube several times. 4. Incubate the samples overnight (or 5-24 hours) at 50ºC. If possible occasionally mix the samples by inverting the tubes make sure the lids are snapped shut as they can sometimes pop loose when warm). [If you are in a hurry you can add more proteinase-k to speed up tissue digestion and reduce the incubation time to 2-4 hours]. 5. Add µl of 6 M NaCl. Shake the samples vigorously for 20 seconds. [do not shake too roughly or you may damage your DNA, but shake harder than simply inverting the tubes]. 6. Microfuge the samples at full speed (12-14,000 rpm) for 5-10 minutes at room temperature. [label new tubes whilst centrifuging samples] 7. Remove supernatent to a new, labeled 1.5 ml microcentrifuge tube. [be very careful not to transfer any of the tissue debris to the new tube unless there is very little tissue it is better to leave a little supernatent rather than transfer the cell debris]. 8. Add an equal volume (~ 800 µl) of cold 100 % ethanol and gently mix by inverting the tube a couple of times you should see white DNA precipitate out of solution. [better yields are achieved if the ethanol is kept at -20ºC before use and then in an ice bucket when on the bench]. [More DNA can be obtained if the samples are left at 20ºC for a few hours or overnight]. 9. Centrifuge the sample at full speed (12-14,000 rpm) for minutes at 4ºC. [room temperature will suffice if there is plenty of DNA]. TIP: When pelleting the DNA, it is very helpful to place the hinge of the microcentrifuge tube towards the outside of the rotor. The subsequent washing steps are made much easier since the DNA pellet can be readily found by looking at the bottom of the centrifuge tube below the hinge. Notes: This method works well from fresh tissue or tissue that has been stored in absolute ethanol at room temperature. If the tissue has been stored in ethanol, place it on some sterile tissue paper and drain off any excess ethanol before proceeding to step 1. 2

4 10. Pour (or pipette) off the supernatent, taking care not to dislodge the pellet of DNA. 11. Wash the DNA pellet in µl of 100 % ethanol (add ethanol, close cap of tube and invert gently, or gently roll the tube on its side). Pour (or pipette) off the ethanol and briefly spin the samples to keep the pellet at the bottom of the tube. 12. Wash DNA pellet with 70 % ethanol as above. After removing the 70 % ethanol, briefly centrifuge the samples to get the last of the ethanol to the bottom of the tube; pipette off the remaining ethanol. 13. Leave the sample to air dry usually min depending upon the temperature. [Do not allow to dry out too much or the DNA will be hard to re-suspend, however, any ethanol left in the tube will inhibit subsequent PCR]. 14. As soon as the sample is just dry, re-suspend the DNA µl (or more f the pellet is large) of sterile distilled water or Tris-EDTA. Notes: 3

5 APPENDIX: Solution recipes: TNES buffer (Tris, NaCl, EDTA, SDS): 10 mm Tris, ph mm NaCl 100 mm EDTA 0.6 % SDS 6 M NaCl is saturated salt solution stored at 37ºC: weigh out 6 M NaCl and heat the solution until it dissolves. Leave to cool at room temperature some crystals will form, this is normal! Do not worry! 4