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Notes
Melting curve analysis is done using a Roche Light Cycler 480.
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 Tm = 88.7°C +/- 1.0°C
Wild type Tm = 83.1°C+/- 1.0°C
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR0160 | TCT GGA CGA AGA GCA TCA GGG | Mutant Forward | A | neo | ||
| oIMR0184 | TAT CAG TCT CAG TGG GGG TG | Common | A | |||
| oIMR0185 | CTG AGC TCT GTT TTC GTC TG | Wild type Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR0160 | 0.50 uM |
| oIMR0184 | 0.50 uM |
| oIMR0185 | 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 |
|---|---|
| 002087 | B6.129P2-B2mtm1Unc/DcrJ |
| 002420 | C.129P2(B6)-B2mtm1Unc/J |
| 005356 | NOD.129(B6)-B2mtm1Unc Ciitatm1Ccum/BhsJ |
| 010565 | NOD.129P2(B6)-B2mtm1Unc Tg(HLA-A24/H2-D/B2M)3Dvs/J |
| 002309 | NOD.129P2(B6)-B2mtm1Unc/J |
| 027849 | NOD.Cg-B2mtm1Unc Tg(B2M)55Hpl Tg(HLA-A*1101/H2-Kb)#Sette/TdilJ |
| 008542 | NOD.Cg-B2mtm1Unc Tg(GFAP-B2m)9Mdos/J |
| 006611 | NOD.Cg-B2mtm1Unc Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ |
| 004548 | NOD.Cg-B2mtm1Unc Mcph1Tg(HLA-A2.1)1Enge Tg(B2M)55Hpl/DvsJ |
| 030547 | NOD.Cg-B2mtm1Unc Prkdcscid H2dlAb1-Ea Il2rgtm1Wjl/SzJ |
| 010636 | NOD.Cg-B2mtm1Unc Prkdcscid Il2rgtm1Wjl/SzJ |
| 018907 | NOD.Cg-B2mtm1Unc Tg(B2M)55Hpl Tg(HLA-B*0702)#Chmb/TdilJ |
| 002570 | NOD.Cg-Prkdcscid B2mtm1Unc/J |
| 13 strains use this protocol | |