Extraction Of DNA From Mouse Tails
Approx. time 1 hour
List of things needed for extraction
— # tails X 4 = # of microfuge tubes
— 10 mg/ml proteinase K or 20 mg/ml of proteinase. Make sure the proteinase K is completely thawed, and mixed well before adding it to the sample.
— 50 mM Tris (pH=8.0), 100 mM EDTA, 0.5% SDS (Proteinase Lysis buffer for tail DNA)
— Phenol/Chloroform (1:1) (It is kept in #1 refrigerator door)
— Chloroform/IAA (CHCl3:IAA @ 24:1) (It is kept in the flammable cabinet)
— 100% Isopropanol
— 70% Ethanol
1. Always keep the tubes in the same order.
2. Always move tubes to a new row after adding each component.
3. When centrifuging always place the microfuge tubes in the centrifuge in the same orientation (with the hinge pointing outward) so you know where the pellet is formed.
Make sure that you know what the identifying number of each mouse is before you cut the toes.
1. Cut mice when they are 8 to12 days old. Cut 1-1.5 cm of tail and place it in a 1.5 ml microfuge tube. The mice do not have teeth at this point and therefore will not bite. Hold the mouse firmly by the back of his neck and the tail between the your pinky and ring finger to prevent the mouse from moving around a lot. Be sure to use sharp scissors so the cut will be clean and done quickly.
2. Add 450ul of Lysis Buffer [50 mM Tris (pH=8.0), 100 mM EDTA, 0.5% SDS] to the Microfuge tube.
3. Next add 25ul of 10 mg/ml proteinase K to the microfuge tube.
4. Incubate microfuge tube at 55 deg C overnight on a rotator located in the lab. If the tail is not completely dissolved the next day add about 15ul of 10 mg/ml proteinase K to the microfuge tube and allow tube to rotate for a couple more hours until the tail is completely dissolved.
(From this point on gloves should be worn at all times and great care must be taken to prevent any of the DNA from being contaminated or mixed up.)
5. Remove the tubes from the rotator and spin the microfuge tubes down for 3 to 4 min to allow the hair to form into a pellet. Pour off the supernatant into a new labelled 1.5 ml microfuge tubes.
6. Add equal volume of 100% Isopropanol. Gently shake the tubes until the DNA clump is seen suspended in the isopropanol.
7. Centrifuge for 1 min. at the highest speed @ room temperature.
8. Pour off the isopropanol. Be careful not to disturb the DNA which is the pellet at the bottom of the tube.
9. Centrifuge for 10 secs at the highest speed @ room temperature if the DNA clump is no longer stuck to the wall of the microfuge tube. Be careful not to disturb the DNA pellet.
10. Air dry the DNA for 1 min.
11. Add 200 ul of SDDW and place the tubes at 45 deg C water bath overnight for approximately 18 to 20 hours. After incubation, tap tube gently to allow DNA to go into solution.
12. Add equal volume of phenol to the microfuge tubes. Shake vigorously for 10 sec
Please note that you cannot store phenol or chloroform in the 15 ml Polystyrene Falcon conical tubes (Falcon #352099) which are hard and clear. They will dissolve through the tubes. They can be temporarily stored in the 50 ml Polypropylene Falcon conical tubes (Falcon # 2070) which are soft and frosted. Remember to discard excess phenol and chloroform in proper waste bottles.
13. Centrifuge for 10 min at the highest speed @ room temperature.
Transfer the upper phase to new 1.5 ml centrifuge tubes using the pipetman. Be careful not to pick up the phenol or the interphase layer of protein by gently and carefully pulling out the supernatant along the walls of the microfuge tube.
14. Add equal volume of Chloroform (CHCl3:IAA @ 24:1) to the microfuge tubes.
15. Centrifuge for 10 min at the highest speed @ room temperature.
16. Pipet out the upper phase, which contains the DNA, and put it into new microfuge tubes. Be careful not to disturb the lower layer of Chloroform.
17. Store purified DNA at 4 deg C.