Stock Number: 006130 Strain Name: CBACa.Cg-ScribCrc/RachJ

Allele: D15Mit93_marker Protocol Name: 006130 MIT markers Method: Standard PCR Version: 2.1 Created: 03-December -2008 Updated: 24-September-2009

Notes: THIS STRAIN NEEDS TO BE TYPED FOR TWO MIT MARKERS LISTED AS REACTION A AND REACTION B. FML Buffer: 500 mM KCl, 100 mM Tris HCl pH 8.3, 15 mM MgCl2, 0.01% Gelatin Add 42 µl of loading buffer (12% ficoll 400, 0.2% bromophenol blue, 0.04 M EDTA) diluted 1:4 with TEN (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10 mM NaCl) to PCR reaction. Load 5 µl on the gel. PCR products are separated on 3.5 % MetaPhor agarose gel with 0.5 x SYBR Green I Nucleic Acid Stain. Expected Results: D15Mit68: Mutant (C57BL/6J) ≈ 112 bp, Wild type (CBA/J) ≈ 120 bp D15Mit93: Mutant (C57BL/6J) ≈ 146 bp, Wild type (CBA/J) ≈ 154 bp

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type D15Mit68-L - TTC CAT GTG AGT TCC AAG CA - - D15Mit68-R - GAA CTG CCA TTC AGA ATA TTT GG - - D15Mit93-L - AAG AAT TGG GGT GGG TAA GG - - D15Mit93-R - CAT GTG CAG AAT ACT TAC ACA TAT GC - -

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 8.12 - 8.12 10 X FML Buffer 1.31 1.09 X 1.31 2.5 mM dNTP 1.04 0.22 mM 1.04 6.6 uM D15Mit93-L 0.24 0.13 uM 0.24 6.6 uM D15Mit93-R 0.24 0.13 uM 0.24 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA (crude prep diluted 1:1 in water) 1.00 - 1.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 55 35 sec - 4 72 30 sec repeat steps 2-4 for 38 cycles 5 72 7 min - 6 10 - hold

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 8.12 - 8.12 10 X FML Buffer 1.31 1.09 X 1.31 2.5 mM dNTP 1.04 0.22 mM 1.04 6.6 uM D15Mit68-R 0.24 0.13 uM 0.24 6.6 uM D15Mit68-L 0.24 0.13 uM 0.24 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA (crude prep diluted 1:1 in water) 1.00 - 1.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 006130 CBACa.Cg-ScribCrc/RachJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling  None found.