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Stock No: 029233
Protocol 35759: Sanger sequencing Assay - Tyr<c-2J>-SEQ
Version 1.0
Notes
Mut = T
WT= G
>chr7:94641468-94641778 311bp AGAACTTGTTGGCAAAAGAATG CTTTTCGGAGACACTCAAATCA
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
ATGTTCTTGGCTGTTTTGTATTGCCTTCTGTGGAGTTTCCAGATCTCTGATGGCCATTTTCCTCGAGCCTGTGCCTCCTCTAAGAACTTGTTGGCAAAAGAATGCTGCCCACCATGGATGGGTGATGGGAGTCCCTGCGGCCAGCTTTCAGGCAGAGGTTCCTGCCAGGATATCCTTCTGTCCAGTGCACCATCTGGACCTCAGTTCCCCTTCAAAGGGGTGGATGACC[g/t]TGAGTCCTGGCCCTCTGTGTTTTATAATAGGACCTGCCAGTGCTCAGGCAACTTCATGGGTTTCAACTGCGGAAACTGTAAGTTTGGATTTGGGGGCCCAAATTGTACAGAGAAGCGAGTCTTGATTAGAAGAAACATTTTTGATTTGAGTGTCTCCGAAAAGAATAAGTTCTTTTCTTACCTCACTTTAGCAAAACATAC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46959 | AGA ACT TGT TGG CAA AAG AAT G | Forward | A | |||
| 46960 | CTT TTC GGA GAC ACT CAA ATC A | Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 46959 | 0.50 uM |
| 46960 | 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
| Stock Number | Strain Name |
|---|---|
| 036977 | B6(Cg)-Dbpem1Drw Tyrc-2J/J |
| 036153 | B6.Cg-Gt(ROSA)26Sorem7Mvw Tyrc-2J/MvwJ |
| 035186 | B6.Cg-Htra3tm1Sud Tyrc-2J/J |
| 029233 | B6.Cg-Kittm1.1Sraf/Mmjax |
| 033605 | B6.Cg-S1pr1tm4.1Rlp Gt(ROSA)26Sortm2.1(luc*/Arrb2*)Rlp Tyrc-2J/J |
| 023428 | B6;129X1-Tyrc-2J Cdkn1atm2Hpw/J |
| 027662 | C57BL/6-Tyrc-Brd Tg(Gh1-luc/EGFP)D8Mrln/J |
| 026622 | NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ |
| 8 strains use this protocol | |