Imran Mungrue imran.mungrue@utoronto.ca

RESUME

METHODS

1) Tail PCR

*Aliquot 3ul ProteinaseK (Gibco 255-30015, 20mg/ml) and 17ul Tail Tip Buffer for each mouse into a 1.5ml eppendorf tube.  *Cut 5mm from distal end of tail  and add to ProteinaseK solution (2-3mm diameter tissue from ear punch can also be used). *Diggest 3hrs-O/N at 55degC with vigorous shaking.  *Add 480ml sterile water, vortex well to mix. *Heat at 100degC for 5min., to degrade ProteinaseK. *Vortex again to mix. *Spin down to pellet hair and bones. *Use 2ul liquid for PCR as follows (1.5uM MgCl2 final conc). gDNA                    2ul 10X PCR Buf         2.5ul 10mM dNTP          0.5ul 25uM pF                0.5ul 25uM pR                0.5ul Taq Pol (5U/ul)        0.1ul Sterile ddWater      18.9ul Tail Tip Buffer: Working Conc                         Preparation 50mM tris pH 8                      2.5ml 1M Tris pH8 20mM NaCl                           200ul 5M NaCl    1mM EDTA pH 8                   100ul 500mM EDTA pH8 1% SDS                                 5ml 10% SDS Sterile ddWater                          42.2ml                                              Autoclave 20min.

Human Specific iNOS gPCR/RT-PCR (450bp product) (Does not amplify murine iNOS) pF TGG CTG GTA CAT GGG CAC AGA G pR TCT TGG GTC TCC GCT TCT CGT C                                30cycles                       ---------------------------------- 94degC   94degC     57degC     72degC     72degC   4degC 2min        30sec        30sec       30sec        4min        00