Preparation of genomic DNA from mouse tails

6/8/04

 

  1. Cut about 0.5cm of tail from a mouse at P10 (or CRL at P21). Place in an eppendorf tube with 0.5mL 70% EtOH and fix for 20Õ to O/N.

 

  1. Aspirate EtOH. Add 300uL cell lysis buffer with 3uL proteinase K per tube. (Proteinase K is stored in undercounter frige.) Use filter tips for all steps if DNA will be used for PCR genotyping. Lyse O/N with shaking at 55C. If you will not process the DNA immediately, store at 4C.

 

GENOTYPERS: You will generally find the tails lysed at this step in the the 55 degree shaker in the warm room. A note explaining which tails to process and how to do the PCR should be left on the clip over my bench.

 

  1. Warm DNA to RT. Add 1.5uL RNAseA to each tube, changing tips in between samples. (RNAseA is stored in the undercounter frige). Invert the tubes 25X to mix solution. Incubate in beads in the warm room at 37C for 15Õ (or up to 60Õ).

 

  1. Cool sample to RT for 1Õ on ice. Add 100uL protein precipitation solution to each tube. Vortex at high speed for >20 seconds to thoroughly mix. Chill on ice for 5Õ. Spin max speed for 3Õ to precipitate proteins. If any residual protein in supe, vortex, chill, and spin again. Do not pour protein into the next tube.

 

  1. Decant supe into a fresh Eppendorf tube containing 300uL 100% isopropanol. Carefully label the tubes with the number for each tail. Invert the tubes 50X to mix solution.

 

  1. Spin top speed for 3Õ to precipitate DNA. A small white pellet should be visible. Pour off isopropanol and dry tubes upside down on paper towel for 5Õ.

 

  1. Add 300uL 70% EtOH to each tube and invert tubes a few times to wash DNA. Spin 3Õ at top speed to anchor pellet.

 

  1. Pellet may be very loose, so do not let tubes sit after spin and carefully pipette off EtOH. Air dry on paper towel for 15Õ. If all EtOH is not gone (and if you can see the pellet), aspirate or pipette out the excess carefully avoiding the pellet.

 

  1. Once all EtOH is removed, add 50uL DNA hydration solution. Hydrate DNA for 1 hour at 65C. Tap tube periodically to help resuspend DNA.  Spin down evaporated solution and gently pipette DNA to fully resuspend. Optional leave DNA O/N at RT to resuspend. Store at 4C. On spec you expect 300-600ug/mL (15-30ug DNA total).

 

 

 

 

 

 

 

 

Buffers:

 

Tail Lysis Buffer:                                            for 500mL

            25mM TrisCl pH 8.0                          6.25mL 2M Tris pH8.0

            10mM EDTA pH 8.0                          10mL 0.5M EDTA pH8.0

            1% SDS                                              25mL 20% SDS

 

Proteinase K:

            Qiagen cat# 19133 (stable at RT)

            >600mAU/mL (about 20mg/mL)

 

RNAseA

            Dissolve to 4mg/mL in ddH20. Boil 15Õ to kill DNAses. Store 4C.

            Roche cat# 109 126

 

Protein Precipitation Solution

            7.5M Ammonium Acetate

                        289.05g Ammonium acetate in 150mL ddH2O, make to 500mL

 

DNA hydration solution (TE)

            10mM Tris pH 7.4

            1mM EDTA pH 8.0

 

 

 

 

If in doubt, remember

(final concentration)(final volume) = (initial concentration)(initial volume)