For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 003709
Protocol 23870: Standard PCR Assay - Tg(Thy1-GFP)
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
Tg tm=89
IPC tm=83.5
Tg= ~300bp
IPC=200bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR7303 | TCT GAG TGG CAA AGG ACC TTA GG | Transgene Forward | A | |||
| oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control | A | |||
| oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control | A | |||
| oIMR9296 | CGC TGA ACT TGT GGC CGT TTA CG | Transgene Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR7303 | 0.50 uM |
| oIMR8744 | 0.50 uM |
| oIMR8745 | 0.50 uM |
| oIMR9296 | 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.