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Stock No: 003342
Protocol 25006: Separated PCR Assay - Hbb<tm1Tow>alternate3
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
Mutant = ~332 bp
Heterozygote = ~332 bp and 490 bp
Wild type = 490 bp
Sequence
Mutant sequence, including the junction:
(gagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggggggaacttcctgactaggg
gaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctaccggtggatgtggaatgtgtgcgag
gccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgcatgctccagactgccttgggaaaagcgcctc
ccctacccggtagaattcctgcagcccggg)GGATCCTGAGAACTTCAGGGTGAGTCTGATGGGCACCTCC
TGGGTTTCCTTCCCCTGGCTATTCTGCTCAACCTTCCTATCAGAAGGAAAGGGGAAGCGATTCTAGGGAGCA
GTCTCCATGACTGTGTGTGGAGTGTTGACAAGAGTTTGGATATTTTATTCTCTACTCAGAAT
CGCTGCTCCCCCTCACTCTGTTCTGTGTTGTCAT
WT Hbb, including junction:
(ctgctggttgtctacccttggacccagcggtactttgatagctttggagacctatcctctgcctctgctatcatgggtaatgccaaagtgaaggcccatggcaagaa
agtgataactgcctttaacgatggcctgaatcacttggacagcctcaagggcacctttgccagcctcagtgagctccactgtgacaagctgcatgt)GGATCC
TGAGAACTTCAGGGTGAGTCTGATGGGCACCTCCTGGGTTTCCTTCCCCTGGCTATTCTGCTCAACC
TTCCTATCAGAAGGAAAGGGGAAGCGATTCTAGGGAGCAGTCTCCATGACTGTGTGTGGAGTGTTG
ACAAGAGTTTGGATATTTTATTCTCTACTCAGAATCGCTGCTCCCCCTCACTCTGTTCTGTGTTGTCA
T
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 12300 | AGA ATA GCC AGG GGA AGG AA | Mutant Reverse | B | Hbb-b1 | ||
| 12316 | CTG TCC ATC TGC ACG AGA CT | Mutant Forward | B | PGK R | ||
| 13545 | ACC CAC AGC CAA ACA CTA GG | Wild type Forward | A | |||
| 13546 | GAA GGG AAG AAG CAA CAT GC | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 13545 | 0.50 uM |
| 13546 | 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.
Reaction B
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 12300 | 0.50 uM |
| 12316 | 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.
Strains Using This Protocol
This is the only strain that uses this protocol.