For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 021504
Protocol 27303: Standard PCR Assay - Tg(Vil1-cre)1000Gum-Alternate 1
Version 2.2
Notes
This assay CANNOT tell the difference between tg/0 and tg/tg.
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
TG= ~150 bp
Hemizygous = ~150 bp and 182 bp
IPC = 182 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 16775 | GCC TTC TCC TCT AGG CTC GT | Common | A | Vil1 | ||
| 16776 | TAT AGG GCA GAG CTG GAG GA | Wild type Reverse | A | Vil1 | ||
| oIMR9074 | AGG CAA ATT TTG GTG TAC GG | Transgene Reverse | A | Cre R |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 16775 | 0.50 uM |
| 16776 | 0.50 uM |
| oIMR9074 | 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.