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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 = 378 bp
Heterozygote = 248 bp and 378 bp
Wild type = 248 bp
Heterozygote = 248 bp and 378 bp
Wild type = 248 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR7720 | GGG ATG CCA CTG CCC TG | Wild type Forward | A | |||
| oIMR7721 | CGA GCC TGA GAT TGT CAA GTG TAT T | Common | A | |||
| oIMR7722 | GGA ATT CCC AGT GAA GGG C | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR7720 | 0.50 uM |
| oIMR7721 | 0.50 uM |
| oIMR7722 | 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 |
| 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 |
| 003986 | MRL.129X1-Fcgrttm1Dcr/DcrJ |
| 003988 | NOD.129X1-Fcgrttm1Dcr/DvsJ |
| 003989 | NZM2410.129X1-Fcgrttm1Dcr/DcrJ |
| 003990 | SJL.129X1-Fcgrttm1Dcr/DcrJ |
| 19 strains use this protocol | |