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Stock No: 017949
Protocol 26880: Standard PCR Assay - Tg(CD207-Dta)1Dhka
Version 3.2
Notes
Validation/LC Conversion data attached to rec 159879
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
65-60
MCA Preferred
Wt = 89°
Mut = 83°
Transgene = 271 bp
Internal positive control = 297 bp
This protocol is for Melt curve- if running on a gel please use the strandard PCR protocol.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 14478 | GAG GCA AAT GAT TGG CAT TCT AC | Transgene Forward | A | |||
| 14479 | CTG GAA AAT TCA AGA AGA GCC T | Transgene Reverse | A | |||
| oIMR9020 | AAG GGA GCT GCA GTG GAG TA | Wild type Forward | A | |||
| oIMR9021 | CCG AAA ATC TGT GGG AAG TC | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 14478 | 0.50 uM |
| 14479 | 0.50 uM |
| oIMR9020 | 0.50 uM |
| oIMR9021 | 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.