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Notes
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Mutant = 174 bp
Heterozygote = 174 bp and 209 bp
Wild type = 209 bp
This assay can distinguish hemi from hom- integration site is known to be on mouse Chr 15.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 35520 | AAG CAC TGG CCT GAG ATA CG | Wild type Forward | A | |||
| 35521 | TTC TGT GAT GAG TCT TTC CTC A | Common | A, B | |||
| 35522 | TTC CCA GAA CTG ACA GCA GA | Mutant Forward | B | H2-D1 |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 35520 | 0.50 uM |
| 35521 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Reaction B
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 35521 | 0.50 uM |
| 35522 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 006611 | NOD.Cg-B2mtm1Unc Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ |
| 035844 | NOD.Cg-Flt3em1Akp Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav Tg(HLA-A/H2-D/B2M)1Dvs/J |
| 039054 | NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv/DvsJ |
| 014570 | NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)1Dvs/SzJ |
| 035855 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)Dvs Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/J |
| 031433 | NOD/ShiLtDvs-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ |
| 031856 | NOD/ShiLtDvs-H2-K1d-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ |
| 7 strains use this protocol | |