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Stock No: 002561
Protocol 17822: Sanger sequencing Assay - Nek8<jck>
Version 2.0
Notes
Mut = TGT
WT= GGG
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
GACATCAGCCAGGTGGGTGGTCCCCGTACCCTGGAGAGGGGAAGTGAGCGCAACTCC
CAGGCTGCACCTGCGCTGGGTTAGATCGGGGAAGTACTGGAAGCTTCCTTCCCCAGTG
GCTTCCCACCTGTTGCTGTTTCTGCCGCACTGTGCCACTGTGCGTGCGGTGGGTGTGGT
TGGAGTTAAAGAGACAGTTACCTTCTGAGAATTTGCAGCCTCCAACTTCCGGCTTGCA
GGGGAGGGGACTCAGACCATGGTAGGAGGCAAATCCTTCACTTTTTTTTTTTCTTTCT
CTAGCCCACCATTGTAGAAGCCTTGCT(g/t)G(g/t)CTATGAGATGGTGCAGGT
GGCCTGTGGGGCCTCTCATGTGCTGGCCCTGTCCACAGATGGAGAGTTATTCGCCTGG
GGCAGAGGAGATGGTGGTAAGCCCACTGCCCAGCTCCAGTGTTTAGGGTCCCTCTCA
CAACTTCTCCATCATGCTTTGTTCATTCCTATACCTGTCTACCTCTCCGTGGCCCATTCT
TTCCTTCCCCCCCTTTTTTTTTCTCGAATTCTATTCATTCATTGAACGAACCATATTTCAT
ACTTTATTAGTGCTCAGATAAGATAAATGAGGTGTTGGCACATTGCTTATCTATTTTTGCAA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 25278 | TTT TCT TTC TCT AGC CCA CCA | Forward | A | |||
| 25279 | TAA GCA ATG TGC CAA CAC CT | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 25278 | 0.50 uM |
| 25279 | 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 | -- | -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.