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Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Conversion/LC Validation data attached to rec 161125
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 = 213 bp
Internal positive control = 415 bp
Transgene Tm = 83.5
IPC Tm = 89
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 13582 | CCA CCA CTT CCT GCC TAC AT | Transgene Forward | A | |||
| 13583 | CCA GAC CGT CTC CTT CTT TG | Transgene Reverse | A | |||
| 21238 | CTG TCC CTG TAT GCC TCT GG | Internal Positive Control Forward | A | |||
| 21239 | AGA TGG AGA AAG GAC TAG GCT ACA | 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 |
| 13582 | 0.50 uM |
| 13583 | 0.50 uM |
| 21238 | 0.50 uM |
| 21239 | 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 |
|---|---|
| 030434 | NOD.Cg-H2-Ab1b-tm1Doi Tg(HLA-DRA,HLA-DRB1*0301)#Gjh/DvYtomJ |
| 021885 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl/SzJ |
| 017637 | NOD.Cg-Prkdcscid Il2rgtm1Wjl H2-Ab1b-tm1Doi Tg(HLA-DRB1)31Dmz/SzJ |
| 029295 | NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(H2-Ea-HLA-DRB1*0401*)1Dv/SzJ |
| 4 strains use this protocol | |