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Stock No: 026816
Protocol 31299: Standard PCR Assay - Igs2<tm1(CAG-xstpx-cas9)Mmw>
Version 2.0
Notes
Common primer: correct is 23906 see REQ 433407 SAC 11-2-17
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 = ~400 bp
Heterozygote = ~400 bp and 373 bp
Wild type = 373 bp
Sequence
Wt Sequence: cagcttcagcctgaagagtaagtagttctctattggcagtttgacacatcctgcccttaccttactaccactgttggctcagcagacacccaggataagtgcactagtgttcctttcctgaccagtgggactgctttttccagATTCCtcttgaagctgaggaggcagtgagagcatgatggcatctaatgagcttggaagtaccagactgccctgatccacagccaggttttgctgaaaagtgaccagtttgtcctcctccagtagagtgggcagctgaagGATTATAatctactgtcaagacttggaggcccctgcagtcaaagtccaatagaatattatgaaatggagaatggcttattttaatctctatagtggaatt
Mutant Sequence: CAGCAGACACCCAGGATAAGTGCACTAGTGTTCCTTTCCTGACCAGTGGGACTGCTTTTTCCAGATTCCTCTTGAAGCTGAGGAGGCAGTGAGAGCATGATGGgcgcgccccttactaggcggccgcgaagttcctataccttttgaagaataggaacttcggaataggaacttcgtcgacaaaggatccgtcggcgaccctacgcccccaactgagagaactcaaaggttaccccagttggggcactactcccgaaaacgtcggcgaccctacgcccccaactgagagaactcaagggcacgccctggcacccgcaccgcggcttcgagaccgtgacctac
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 23906 | TGG CAG TTT GAC ACA TCC TG | Common | A | |||
| 23907 | ATC TGG GGC CAT AAA TGC T | Wild type Reverse | A | |||
| 23908 | CCG CCA CTC GAC GAT GT | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 23906 | 0.50 uM |
| 23907 | 0.50 uM |
| 23908 | 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.