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Stock No: 029295
Protocol 19705: Standard PCR Assay - Tg(HLA-A2/H2-D/B2m)
Version 2.0
Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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
Transgene = ~200 bp
IPC = 324 bp
Tg Tm = 91
IPC Tm = 83
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 10647 | CTT CAT CGC AGT GGG CTA C | Transgene Forward | A | |||
| 10648 | CGG TGA GTC TGT GAG TGG G | Transgene Reverse | A | |||
| oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
| oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 10647 | 0.25 uM |
| 10648 | 0.25 uM |
| oIMR7338 | 0.50 uM |
| oIMR7339 | 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 |
| 029295 | NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(H2-Ea-HLA-DRB1*0401*)1Dv/SzJ |
| 031856 | NOD/ShiLtDvs-H2-K1d-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ |
| 4 strains use this protocol | |