www.duke.edu/~yzhuang

PCR Genotyping Protocol

1) Cut toes of mice at 7-12 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 50oC for >3hrs or O/N.

            Lysis Buffer: 100mM NaCl, 10mM Tris pH8.0, 25mM EDTA, 0.5% SDS,
            add fresh 0.1mg/ml Proteinase K (100X, 10mg/ml in ddH2O stock)

 

3) Extract protein with phenol once and chloroform once.  Precipitate DNA with 2 volumes 95% EtOH and spin 5min.  Remove sup. and wash once with 70% EtOH.  Quick spin tube, remove any extra liquid and add 0.5ml TE Buffer or ddH2O.

 

4) Prepare a cocktail of PCR reagents for each strain of mouse to be analyzed. For primer sequences click here.

           Cocktail (for 10 reactions): 92.5ul ddH2O, 15ul 10X PCR Buffer, 15ul DMSO,
           15ul 10mM dNTP, 5ul primer #1(10uM stock), 5ul primer #2 (10uM stock),
           2.5ul homemade TAQ polymerase

           10X PCR Buffer: 166mM Ammonium Sulfate, 670mM Tris pH8.8, 67mM MgCl2, 50mM 2-Me,            67uM EDTA

*Id3ko genotyping* - Add additional 3.1 ul of 50mM MgCl2 to PCR cocktail


5) Use 14ul cocktail + 1ul toe DNA for each PCR reaction

            PCR program (YZ1):

            1) 93oC - 1.5 min (initial denature)

            2) 93oC - 0.5 min (denature)

            3) 57oC - 0.5 min (anneal)

            4) 65oC - 3 min (extend)

            5) Goto step #2 39 times.

            approximate running time is 3.5hrs with MJ thermal cycler.


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