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Stock No: 002703
Protocol 17644: Sanger sequencing Assay - Hydin<hy3> SEQ
Version 1.0
Notes
Mut = CCC-
WT= CCCC
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
AACCTCCACGACTAAATGCATGATAAATACTTTAGTTAAGTCATGAATGCATGAAGTGT
ATGTGTTGGTGTATGACTGGGGATTTAAGGAGCTAACAGGGAATGGATATAAGCAGAG
GGAATGACACACATCTGTGAAGATAAAGATGTCACAGTGAGACCCTAGTTTGTATATA
CATTAAACATAGTTTTAAAGAGGGCCTGGGGATGAGGCTGAGACACTGAAGTGCCAG
GAGGGCAATGGAACCTGAGATTGATCGACCGGAACCCACGTGAACTATCTGGACGCA
GTGGGGCATACTTAAGATACCAAATTTGGAAAGGGAGAGAGACAGGGGTCCTTGAGT
TTTGCTGGATATCCAGTTTCCCCCAACCAGGAAACCCCAGGCTCAGTGAAAGATCCT
GAATCAAAAAACTAAAGTGTATAGAGGAAAACACCTGATATTGAGCTCTGACCTGTA
CGCATACACCCACCACATTAAATAAATAAATAAATAAATAAATAAATAAATAAATAAAT
AAATAAAATACACACATACACATGCATGCACATGCATACACATACATACATATACATAC
ATATAAATACATATACATACATACTTATTTACTTACTTACTTAAGACAAGTTAGAAAGTA
ATGAGACATAGTAATGCATTATATATACAATCCTAAGTCATTGCCTTAGGATTTGTTGAT
CAAGGGAGGCTACTGGGGGAAAGAAGTCTTTCTCACCTCAAATTTTATCTTCCAGGA
TTT(cccc)ATACCTTGATGTGTTCCCTCAATAATACATCTTTGGTCCCCATGACCTTCAAA
CTGCGTGTCCGCGGCGATGGCGAAGGCATGAGTAGCATCCCAAGTTACTCTCAGGAG
TCAGACAGCAAGCAATGGTCCGGGATTAATACAGAAATGCCTACCACAAAACCCAAA
GAATTCACCATCTCTCCCAACAGTGGCACCATCCGTGCCCAGGGCTTTACTGCCATCAAGGTAAAGTGT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 25792 | TGA TCA AGG GAG GCT ACT GG | Forward | A | |||
| 25793 | ACC ATT GCT TGC TGT CTG ACT | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 25792 | 0.50 uM |
| 25793 | 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 | -- | -1.0 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles | |
| 6 | 94.0 | -- | |
| 7 | 55.0 | -- | |
| 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.