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Stock No: 013749
Protocol 29011: Standard PCR Assay - Igs2<tm#ACTB-tdTomato,-EGFP>
Version 2.2
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 = 230 bp
Heterozygote = 230 bp and 323 bp
Wild type = 323 bp
This assay cannot distinguish the Igs<tm1ACTB-tdTomato,-EGFP from Igs<tm2ACTB-tdTomato,-EGFP. To distinguish run in conjuntion with Generic EGFPC-terminus-MADM TG
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 11362 | TGG AGG AGG ACA AAC TGG TCA C | Wild type Reverse | A | |||
| 11363 | TTC CCT TTC TGC TTC ATC TTG C | Wild type Forward | A | |||
| oIMR7320 | TCA ATG GGC GGG GGT CGT T | Mutant Reverse | A | CMV Promoter |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 11362 | 0.50 uM |
| 11363 | 0.50 uM |
| oIMR7320 | 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.