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Stock No: 002521
Protocol 15144: Pyrosequencing Assay - Ighmbp2<nmd-2J>PYRO
Version 2.0
Notes
This genotyping assay uses pyrosequencing technology and is run on the Biotage PSQ 96MA. The Jackson Laboratory is not posting the complete details of our pyrosequencing genotyping assays as the primers for pyrosequencing cannot be used for sequencing using more traditional methods. The wild type and mutant nucleotides and the flanking DNA sequence are provided below.
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 = G/G
Heterozygote =A/G
Wild type = A/A
sequencing primer is on the forward strand
Sequence
gctcctgattttggctctggtcctcacagAGCACTGATGACACTGAAGAAGTACCACTCTGG
GCCAGCATCCTCCCTCATAGACATCCTGTTGGGCAGCTCCACCCCAAGTCCTGCCATGGAAAT
ACgtgaggatttttaattttctc(a/g)gtatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgaga
gagagagagagagagagagagagagagagggagagagagagagagatggagggggaggga
gggagagagagagatggagggggagggagggagagagagagagagaatg
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 15927 | AGT CCT GCC ATG GAA ATA CG | |||||
| 15928 | Fluorophore | GTC CTT CTG ATA TAC CCC AAG CA | ||||
| 15929 | AGG ATT TTT AAT TTT CTC A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTPS-kapa | 0.20 |
| 15927 | 0.50 |
| 15928 | 0.50 |
| Glycerol | 5.00 |
| Kapa 2G HS taq polym | 0.01 |
| 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 | |
| 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.