Stock Number: 005589 Strain Name: NOD.Cg-Prkdcscid H2-Ab1tm1Doi/SzJ

Allele: Prkdcscid Protocol Name: Prkdcscid Method: Restriction Enzyme Digest Version: 1.1 Created: 04-August -2000 Updated: 21-July -2010

Expected Results: Mutant  = 38 bp, 28 bp, and 11 bp Heterozygote = 68 bp, 38 bp, 28 bp and 11 bp Wild type = 68 bp and 11 bp

Gel Image

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Gel Information: Add DNA Loading Dye*** to digested products and separate on a 3% agarose gel.**

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0803 - GGA AAA GAA TTG GTA TCC AC - Forward oIMR0804 - AGT TAT AAC AGC TGG GTT GGC - Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 5.38 - 5.38 10 X AB PCR BufferII 1.20 1.00 X 1.20 25 mM MgCl2 1.20 2.50 mM 1.20 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR0803 0.60 1.00 uM 0.60 20 uM oIMR0804 0.60 1.00 uM 0.60 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 1.5 min - 2 94 30 sec - 3 53 30 sec - 4 72 30 sec repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold**

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) 10 X AB PCR BufferII 1.20 - 1.20 ddH2O 6.30 - 6.30 10 U/ul Alu I 0.50 - 0.50 This genotyping protocol is used for the following strains:   Stock Number Strain Name 001131 C3SnSmn.CB17-Prkdcscid/J 001303 NOD.CB17-Prkdcscid/J 001803 CBySmn.CB17-Prkdcscid/J 001913 B6.CB17-Prkdcscid/SzJ 002038 CB17;HPG-Prkdcscid Gnrh1hpg/Bm 002313 NOD.Cg-Prkdcscid Emv30b/Dvs 002380 NOD.Cg-Tg(Ins2-TAg)1Lt Prkdcscid/DvsJ 002570 NOD.Cg-Prkdcscid B2mtm1Unc/J 002571 NOD.Cg Prkdcscid-B2mb/Dvs 002577 B6;CB17-Ghrhrlit Prkdcscid/Bm 003843 NOD.Cg-Prkdcscid Tg(Ins2-GAD2)1Lt/LtJ 003844 NOD.Cg-Prkdcscid Tg(Ins2-GAD2)2Lt/LtJ 004083 NOD.129(B6)-Prkdcscid Iduatm1Clk/J 004230 NOD.Cg-Prkdcscid Tg(Ins2-E3)1Dvs/DvsJ 004257 NOD.Cg-Prkdcscid Tg(TcrLCMV)327Sdz/Dvs 004262 NOD.Cg-Prkdcscid Tg(HLA-A2.1)1Enge/Dvs 004346 NOD.Cg-Prkdcscid Tg(Ins2-CD80)3B7Flv/DvsJ 004606 NOD.Cg-Prkdcscid H2-Ab1tm1Doi Tg(HLA-DQA1,HLA-DQB1)1Dv/SzJ 004644 NOD.Cg Prkdcscid-Tg(CSF2)2Ygy Tg(IL3)1Ygy Tg(KITLG)3Ygy/YgyJ 004919 B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ 005053 NOD.Cg-Prkdcscid Gusbmps/SndsJ 005345 NOD.Cg-Cd38tm1Lnd Prkdcscid/LtJ 005557 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ 005589 NOD.Cg-Prkdcscid H2-Ab1tm1Doi/SzJ 006605 NOD.Cg-Prkdcscid Emv30b Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ 006609 NOD.Cg-Prkdcscid Tg(HLA-A2.1)1Enge/DvsJ 007840 NOD.Cg-Prkdcscid Tg(Ins2-CD86)12B70Flv/FswJ 010605 STOCK Prkdcscid Gnrh1hpg/BmJ 011066 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CSF2)2Ygy Tg(IL3)1Ygy Tg(KITLG)3Ygy/YgyJGckRolyJ 012478 NOD.Cg-Prkdcscid Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz/GckJ 012479 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz/GckRolyJ 013062 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ 014570 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)1Dvs/SzJ 017619 NOD.Cg-Prkdcscid Tg(CAG-EGFP)1Osb/KupwJ 017620 NOD.Cg-Prkdcscid Tg(CAG-DsRed*MST)1Nagy/KupwJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.