DNA Extraction:

Solution Recipe and Extraction Method

Goals and Rationale:

Extraction solution recipe for use in extracting DNA from mouse tails and ear punches used in mouse PCR genotyping. This recipe makes 200ml of extraction solution. Requires overnight digestion in hot water bath. For quick soft tissue extraction, an Invitrogen kit is available. Also, Sigma sells a rapid DNA extraction kit which may become standard in the lab (see Extract-n-Amp protocol) .

 

 

Safety Concerns

         Caution: This protocol potentially uses biohazardous dry SDS detergent powder- Follow the specific handling procedures and observe MSDS recommendations, wear dust mask and/or use fume hood when weighing powder. Be careful not to spill on balance, and be sure to wipe down the apparatus after measuring as SDS crystals disperse easily. Add Proteinase K in fume hood as this is a potential biohazard as well.

 

Method

 

Tail DNA Extraction:

 

Ø     Thaw appropriate number of aliquots of “tail solution” (extraction solution) stored in 500ml aliquots at –20^oC (see recipe below). Bring to mouse room.

Ø     Immobilize mouse in V-shaped Perspex holder. Cut ~1/2 cm of tail off using a sharp razor blade. Place tail tip in extraction solution, and label appropriately.

Ø     Bring samples back to the lab and place tubes in 55°C waterbath overnight (at least 6-7 hours) until completely digested.

         Note: Some hair may remain undigested at bottom of tube.

 

Ø     Vortex samples to make sure they are digested entirely.

        Note: If tail is not digested completely, add another aliquot of extraction solution and put in water bath for another 3-4 hours.

 

Ø     Microcentrifuge samples at 14.000 rpm for 10 minutes. Meanwhile, add 950ul of ice cold 200 proof ethanol to clean 1.5ml microcentrifuge tubes and label accordingly.

 

 

Ø     After samples finish spinning, transfer 475ul of the supernatant to the appropriate tube containing ethanol.

Note: Remove supernatant slowly as pelleted hair and other debris sits very loosely at tube bottom.

Ø     Vortex sample in ethanol tube, checking to be sure that a DNA clot forms. Clot should look stringy and whitish-colored.

 

Ø     Spin at 14,000 rpm for 10 minutes. Remove ethanol, tip tubes upside down, and dry pellets for at least an hour. Check to see that pellet is visible.

 

Ø     Resuspend pellet in 100ml H2O and store at 4°C until use.

 

Alternatives and Comments

 

Ø     To extract DNA from a 1mm-2mm diameter ear punch, follow the same protocol. You may be able to use a shorter digestion time than overnight.

Ø     The protocol for Extract-n-Amp preparation is in a separate method.

 

Reagents

 

To make up 200ml of extraction solution:

 

     Chemical                            Amount       [Stock]        [Final] (in H2O)

 

1)  TRIS***                               20ml           1M                       100mM

2)  EDTA      2ml            0.5M                     5mM

3)  SDS                  4ml            10%                      0.2%

4)  Proteinase K              100mg           ----                       500mg/ml

5) Sodium Chloride (NaCl)        8 ml             5M                       200mM

 

***Reagent Storage: Tris, EDTA, SDS, and NaCl can all be found on the reagent shelf in 6-256. Proteinase K is in the dessicator box in the -20°C Dewey freezer.

Ø     Dissolve Proteinase K in 150ml of ddH₂O in flow hood.

Ø     Add liquid components. Stir thoroughly until all components are dissolved and well mixed. Increase volume to 200ml final using ddH₂O, using suitable graduated cylinder.

Ø     Place 400 empty 1.5ml microcentrifuge tubes into 4 white styrofoam freezer boxes. Aliquot 500ml of extraction solution into each tube using a repeater pipetman. Set the pipetman to 2 and use a 12.5ml combitip. Store solution at -20°C until use. Label box as “Tail Solution” with date.

 

 

 

Stock Solutions

 

1) TRIS                                   1M                                100mM

Fisher #BP152-5 [crystals; Mol.Wt. = 121.14g].

     Order from Ustores (BP1525)

     (121.14g X 1M X 1 L = 121.14 1L H₂O).

To 800 ml H₂O in Erlenmeyer flask, add 121.14g TRIS, pH to 8.5 with concentrated HCl. Let stir thoroughly and check pH again. Add H₂O to 1 liter.

 

2)  EDTA      0.5M                                 5mM

     Fisher #BP120-500 [crystals:Mol.Wt. = 372.24g]

     Order from Ustores (BP120500)

     (372.24g X .5M X 500 ml = 93.06g for 500 ml of .5M stock)

    

To 400 ml ddH₂O, add 93.06g of disodium EDTA·2H₂O. Stir vigorously on magnetic stirrer and pH to 8.0 with NaOH. Add ddH₂O to 500ml.

 

3)  SDS                  10%                                 0.2%

     Fisher #BP166-100 - [powder, Mol.Wt. = 288.38]

Order from Ustores (BP166100)

     (500 ml X 10% = 50g/500ml)

 

Add 50g of SDS to 400 ml ddH₂O. Heat to ~68° to aid dissolution. Adjust pH dropwise to 7.2 with concentrated HCl. Add ddH₂O to 500ml.

 

4)  Proteinase K              100mg                             500mg/ml

     Sigma #P-6556 [powder, 37U/mg]

Order from Ustores (P6556-100mg)

 

No stock solution. Add vial directly to final solution.

 

5) Sodium Chloride (NaCl)        5M                              200mM

     Fisher #5271-10 [crystals, Mol.Wt. = 58.44g]

Order from Ustores (527110)

(58.44 x 5 x 0.5 = 146.1g for 500ml of 5M stock)

 

Dissolve 146.1g of NaCl in 400ml of ddH₂O. Adjust volume to 500ml with ddH₂O.