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Notes
JR8179 AAAATGACTGAGTATAAACTTGTGGTGGTTGGAGCTG(G/A)TGGCGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCAGAATCACTT
JR33068 ATGACTGAGTATAA(A/G)CTTGTGGTGGTTGGAGC(T/A)(G/T)G(T/C)GG(C/T)- a SphI site is created by these nucleotide changes
JR33316 AAAATGACTGAGTATAA(A/G)CTTGTGGTGGT(T/A)GGAGCT(G/C)G(T/A)GGCGTAGGCAAGAGCGCCTTGA
JR33317 AAAATGACTGAGTATAA(A/G)CTTGTGGTGGTTGGAGC(T/A)GG(T/A)G(G/A)CGT(A/C)GGCAAGAGCGCCTTGACGATACAGCTAATTCAGAATCACTT
JR8179 AAAATGACTGAGTATAAACTTGTGGTGGTTGGAGCTG(G/A)TGGCGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCAGAATCACTT
JR33068 ATGACTGAGTATAA(A/G)CTTGTGGTGGTTGGAGC(T/A)(G/T)G(T/C)GG(C/T)- a SphI site is created by these nucleotide changes
JR33316 AAAATGACTGAGTATAA(A/G)CTTGTGGTGGT(T/A)GGAGCT(G/C)G(T/A)GGCGTAGGCAAGAGCGCCTTGA
JR33317 AAAATGACTGAGTATAA(A/G)CTTGTGGTGGTTGGAGC(T/A)GG(T/A)G(G/A)CGT(A/C)GGCAAGAGCGCCTTGACGATACAGCTAATTCAGAATCACTT
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
Sequence
aaagttgtaagtgacttacaggttactctgtacatctgtagtcactgaattcggaatatcttagagttttacacacaaaggtgagtgttaaaatattgataaagtttttgataatcttgtgtgagacatgttctaatttagttgtattttattatttttattgtaagGCCTGCTGAAAATGACTGAGTATAAACTTGTGGTGGTTGGAGCTGGTGGCGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCAGAATCACTTTGTGGATGAGTATGACCCTACGATAGAGgtaacgctgctctacagtctgcgtgcgcttgtaaaggacggcagccagccgctttgaaaaagatatcatttttatatttatt
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45690 | GGT TAC TCT GTA CAT CTG TAG TCA CTG | Forward | A | |||
| 45691 | GCT GCC GTC CTT TAC AAG C | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 45690 | 0.50 uM |
| 45691 | 0.50 uM |
| Glycerol | 6.50 % |
| 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.