PCR Genotyping Protocol

1) Cut toes of mice at approx. ten days of age and record sex, color, and strain.  Place toes in marked, individual eppendorf tubes.

2) Add 0.1ml of lysis buffer to each tube.   Incubate at 50^oC for >3hrs or O/N.

            Lysis Buffer

            100mM NaCl

            10mM Tris pH8.0

            25mM EDTA

            0.5% SDS

            add fresh 0.1 mg/ml Proteinase K (100X, 10mg/ml in ddH₂0 Stock)

3)  Extract protein with phenol once and chloroform once.  Precipitate DNA with 2 volumes 95% EtOH and spin 5min.  Remove supt. and wash once with 70% EtOH.  Quick spin tube, remove any extra liquid and add 0.5ml TE Buffer.          

4) Prepare a cocktail of PCR reagents for each strain of mouse to be analyzed (appropriate primers are listed separately).

            Cocktail (for 10 reactions)                                           

            92.5 ul  ddH20                                      

            15 ul     10X PCR Buffer

            15 ul     DMSO

            15 ul     10mM dNTP

            5 ul       primer #1 (10 uM stock)

            5 ul       primer #2 (10 uM stock)

            2.5 ul    TAQ polymerase

*Id3^ko genotyping* - Add additional 3.1 ul of 50mM MgCl₂ to PCR cocktail

use 14 ul cocktail plus 1 ul toe DNA for each PCR reaction

            10X PCR Buffer

            166mM Ammonium Sulfate     

            670mM Tris pH 8.8

            67mM MgCl₂

            50mM 2-Me

            67uM EDTA

            PCR program

            1) 93^oC - 1.5 min (initial denature)

            2) 93^oC - 0.5 min (denature)

            3) 57^oC - 0.5 min (anneal)

            4) 65^oC - 3 min (extend)

            5) Goto step #2 39 times.

            approximate running time is 3.5hrs with MJ thermal cycler.

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