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Stock No: 005445
Protocol 13433: Pyrosequencing Assay - Cyba
Version 3.0
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.
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 = C/C
Heterozygote = T/C
Wild type = T/T
Heterozygote = T/C
Wild type = T/T
separated by gel electrophoresis on a 1.5% agarose gel.
Sequence
GGTGCTGTACTGGGCTTGTTACACCTGCAGTTTGGGGACACACCCATATGGCCA
CGCTCCAGCTCGGGCCTTTTCCCCAGCAAGATGTCTGTGGCTTGAATCAAAGGA
CACATCGTCACAAAGAGGAGCTTCTACAAACCCCAAGCTGGACCTGATGAGAGC
TAGCCTCTGCCCATCCCCCGTCCAGGGCCCCGGGGAGGGAATCTCCCTCTGCG
ACTGTGTGACCAGATGCCCACTGACTGCTATCTGTTTGCTTCCTCCTGTCATGCC
TCAAGGTTGTCGGTGCCTGCAGGCTTCCTCCTGGCCACCATCCTGGGGACCGT
CTGCTTGGCCATTGCCAGTGTGATC(t/c)ATCTGCTGGTAAGTGGCTGCCCTCAATGG
TCTGCCTCCCTCCCGCACCCACCCTCATGATCTTGGTGCCCGTGGCTCTCGGAG
CAGCTTTCTCGCTCCCATTGGCTGTGTCGAATGCTCCTCTGCTGTATCCTGGGCA
CTTCCCTGATGAAGCTGTACTGTGGCTCGGAGAAGCAGCGTGGTTGGTCCAGGT
CATACCTCCAAGCCTCCGTGTGGGCCCATGTCTCATCCAAGCCTGCCTGAGTTTG
GGTGGTGTTAGGTGGACTTCCCAGGAGCTCATGACACAGAAGCATCTATTTTATTTATTCTT
CGCTCCAGCTCGGGCCTTTTCCCCAGCAAGATGTCTGTGGCTTGAATCAAAGGA
CACATCGTCACAAAGAGGAGCTTCTACAAACCCCAAGCTGGACCTGATGAGAGC
TAGCCTCTGCCCATCCCCCGTCCAGGGCCCCGGGGAGGGAATCTCCCTCTGCG
ACTGTGTGACCAGATGCCCACTGACTGCTATCTGTTTGCTTCCTCCTGTCATGCC
TCAAGGTTGTCGGTGCCTGCAGGCTTCCTCCTGGCCACCATCCTGGGGACCGT
CTGCTTGGCCATTGCCAGTGTGATC(t/c)ATCTGCTGGTAAGTGGCTGCCCTCAATGG
TCTGCCTCCCTCCCGCACCCACCCTCATGATCTTGGTGCCCGTGGCTCTCGGAG
CAGCTTTCTCGCTCCCATTGGCTGTGTCGAATGCTCCTCTGCTGTATCCTGGGCA
CTTCCCTGATGAAGCTGTACTGTGGCTCGGAGAAGCAGCGTGGTTGGTCCAGGT
CATACCTCCAAGCCTCCGTGTGGGCCCATGTCTCATCCAAGCCTGCCTGAGTTTG
GGTGGTGTTAGGTGGACTTCCCAGGAGCTCATGACACAGAAGCATCTATTTTATTTATTCTT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 8523 | CCA TTG CCA GTG TGA | SEQ | ||||
| poIMR0323 | CTT CCT CCT GGC CAC CAT | Forward | ||||
| poIMR0324 | Fluorophore | ATT GAG GGC AGC CAC TTA CC | Reverse |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTPS-kapa | 0.20 |
| poIMR0324 | 0.50 |
| poIMR0323 | 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.
Strains Using This Protocol
This is the only strain that uses this protocol.