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Stock No: 013167
Protocol 28799: Standard PCR Assay - Ahi1<tm1Jgg & tm2.1Jgg>
Version 2.2
Notes
This assay does not work well without the use of a Hotstart Taq.
Only the two primers 10987/10988 are necessary for genotyping the loxp flanked allele in Stock# 013167. Adding primer 10989 (mutant primer for null strain Stock# 013168) will not confuse loxp fanked genotyping.
Only the two primers 10987/10988 are necessary for genotyping the loxp flanked allele in Stock# 013167. Adding primer 10989 (mutant primer for null strain Stock# 013168) will not confuse loxp fanked genotyping.
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 (loxp flanked) = 399 bp
Heterozygote = 399 bp and 297 bp
Wild type = 297 bp
Heterozygote = 399 bp and 297 bp
Wild type = 297 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 10987 | TTC ATC TGT ATT GAG CCA TTG TC | A | ||||
| 10988 | TGT CTG TGT GTG CTG GGA GA | A | ||||
| 10989 | CAT TCC AGC CAG ATC ACA AA | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 10987 | 0.50 uM |
| 10988 | 0.50 uM |
| 10989 | 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
This is the only strain that uses this protocol.