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Stock No: 027344
Protocol 27239: Sanger sequencing Assay - Gba1<tm1Rlp>
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
Ref Seq: TGGCCAGTGAGAGCACTGACtt/cc
Mut = CC
WT= TT
Sequence
gctcagggctaagagtctggctgtgttgcccagggaagcctggggtcctgatgatttctatcttcacgttcagCAAGTTCATTCCCGAGGGATCCCAGAGAGTGGCGTTGGTGGCCAGTGAGA
GCACTGAC(tt/cc)GGAAACAGTAGCACTGTTACGCCCTGACGGCTCTGCAGTTGTGGTCGTGTTAAACCGgtaagctgggtgcaagggcaggtctgggcctgagccgtgtgagg
tggctggcatcagcaggttgagtcttgacttttcttctctccagATCTTCGGAGGATGTCCCTCTTACCATCAGTGATCCTGACCTGGGCTTCCTGGAGACCGTGTCACCTGGCTAC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 22868 | GCT CAG GGC TAA GAG TCT GG | Forward | A | |||
| 22869 | GTA GCC AGG TGA CAC GGT CT | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 22868 | 0.50 uM |
| 22869 | 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.