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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 = ~90 bp
Heterozygote = ~90 bp and 192 bp
Wild type = 192 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 23256 | AGG CAG GAG GAC CAA CAA G | Common | A | |||
| 25053 | GTA TCT AGA ACC GGT GAC G | Mutant Forward | A | |||
| 25054 | TGT CTG TCG TCT TGG ACT GG | Wild type Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 23256 | 0.50 uM |
| 25053 | 0.50 uM |
| 25054 | 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
| Stock Number | Strain Name |
|---|---|
| 017700 | B6.129-Rag1tm1Mom Fcgrttm1Dcr/DcrJ |
| 003982 | B6.129X1-Fcgrttm1Dcr/DcrJ |
| 021146 | B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(CAG-FCGRT)276Dcr/DcrJ |
| 018441 | B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(FCGRT)32Dcr/DcrJ |
| 018541 | B6.Cg-Fcgrttm1Dcr Prkdcscid/DcrJ |
| 004919 | B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ |
| 014565 | B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ |
| 016919 | B6.Cg-Rag1tm1Mom Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ |
| 037524 | B6.Cg-Tg(ALB)9463J Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr/J |
| 031644 | B6.Cg-Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr Prkdcscid /J |
| 025201 | B6.Cg-Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr/MvwJ |
| 035448 | B6.Cg-Tg(FCGRT)32Dcr Fcgrttm1Dcr Ace2em4Lutzy/J |
| 029686 | B6.Cg-Tg(FCGRT)32Dcr Fcgrttm1Dcr Ighg1em2(IGHG1)Mvw/MvwJ |
| 037043 | B6.Cg-Tg(K18-ACE2)2Prlmn Tg(FCGRT)32Dcr Fcgrttm1Dcr/2J |
| 034902 | B6.Cg-Tg(K18-ACE2)2Prlmn Tg(FCGRT)32Dcr Fcgrttm1Dcr/J |
| 021455 | BXSB.129X1-Fcgrttm1Dcr/J |
| 025099 | BXSB.Cg-Fcgrttm1Dcr Tg(FCGRT)99Dcr/J |
| 003986 | MRL.129X1-Fcgrttm1Dcr/DcrJ |
| 003988 | NOD.129X1-Fcgrttm1Dcr/DvsJ |
| 028615 | NOD.Cg-Fcgrttm1Dcr Prkdcscid Il2rgtm1Wjl Tg(FCGRT)32Dcr/J |
| 028616 | NOD.Cg-Tg(CAG-FCGRT)276Dcr Fcgrttm1Dcr Prkdcscid Il2rgtm1Wjl/J |
| 003989 | NZM2410.129X1-Fcgrttm1Dcr/DcrJ |
| 003990 | SJL.129X1-Fcgrttm1Dcr/DcrJ |
| 23 strains use this protocol | |