For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 003259
Protocol 27729: Standard PCR Assay - Igf1<tm2Ts>alternate1
Version 1.2
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 = 280 bp
Heterozygote = 231 bp and 280 bp
Wild type = 231 bp
This assay will only tell absence or presence of Neo inserted in to Exon 3 (Midi form). A neo qPCR MUST be run along with this assay to determine het from hom for Neo (Midi).
The construct interrupted exon 3 with a neomycin cassette, with a thymidine kinase gene and vector sequences inserted 5' of the endogenous exon 3.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21761 | AGA GGG GAT GGG AGA GCT AC | Wild type Reverse | A | |||
| 24506 | AGG ACC CAG AAG GAT ACC TGA | Common | A | |||
| oIMR8444 | GCC TGA AGA ACG AGA TCA GC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 21761 | 0.50 uM |
| 24506 | 0.50 uM |
| oIMR8444 | 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
This is the only strain that uses this protocol.