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Stock No: 012836
Protocol 35515: Standard PCR Assay - Tg(Thy1-TARDBP)4Singh-Chr6
Version 1.0
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 = 100 bp
Heterozygote = 100 bp and 161 bp
Wild type = 161 bp
>chr6:56525024+56525184 161bp CAATTGGTTCCCAGCTTGTG GGCATTTCAATTCACCAAGG
This assay is capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 6.
This assay is designed around this insertion site, and has been tested on hom animals (DJL 4-17-19).
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46268 | CAA TTG GTT CCC AGC TTG TG | Common | A | |||
| 46269 | GGC ATT TCA ATT CAC CAA GG | Wild type Reverse | A | |||
| 46270 | AGG TTC AGG GGG AGG TGT G | 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 |
| 46268 | 0.50 uM |
| 46269 | 0.50 uM |
| 46270 | 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.