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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 = 215 bp
Internal positive control = 324 bp
IPC Tm = 82 Area = 33.5
Transgene Tm = 79.5 Area = 50 (WT* Tm = 83 Area = 28.5)
*MCA WT with Tg assay @TD50EG but expect minimal -to-no WT @TD60EG (TD60EG MCA plate was not exported). SC 6-28-12
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 14909 | AGA GGG AAG GCC TGA TGT GT | A | ||||
| 14910 | CAA GAT AGA CCA TAT CCT GCA CC | A | ||||
| oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | A | ||||
| oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 14909 | 0.50 uM |
| 14910 | 0.50 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 |
|---|---|
| 023837 | B6.Cg-Tg(SNCA)OVX37Rwm Sncatm1Rosl/J |
| 018442 | C57BL/6N-Tg(SNCA)129Mjff/J |
| 010710 | FVB;129S6-Sncatm1Nbm Tg(SNCA)1Nbm/J |
| 010799 | FVB;129S6-Tg(SNCA*A53T)1Nbm Sncatm1Nbm Tg(SNCA*A53T)2Nbm/J |
| 027227 | STOCK Gba1tm1Rlp Tg(SNCA*A53T)1Nbm Sncatm1Nbm Tg(SNCA*A53T)2Nbm/Mmjax |
| 027344 | STOCK Gba1tm1Rlp Sncatm1Nbm Tg(SNCA)1Nbm/Mmjax |
| 6 strains use this protocol | |