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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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
TG= 88 bp
IPC= 74 bp
Sequence
Tg Sequence:
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52079 | GGA TGG AGT ACC GAC TGG AG | Transgene Forward | A | hACE2 cDNA | ||
| 52080 | CAT ATG CTT TAT CTC CAA GAG CTG | Transgene Reverse | A | hACE2 cDNA | ||
| 52081 | Fluorophore-1 | AGA CCA AAG CAT CAA AGT GAG G | Quencher-1 | Tg Probe | hACE2 cDNA | |
| oIMR1544 | CAC GTG GGC TCC AGC ATT | Internal Positive Control Forward | A | |||
| oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | Internal Positive Control Reverse | A | |||
| TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52079 | 0.40 uM |
| 52080 | 0.40 uM |
| oIMR1544 | 0.40 uM |
| oIMR3580 | 0.40 uM |
| Tg Probe | 0.15 uM |
| IC Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | repeat steps 2-3 for 40 cycles |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 035077 | B6.Cg-Stat1tm1Dlv Tg(K18-ACE2)2Prlmn/J |
| 035041 | B6.Cg-Tg(K18-ACE2)2Prlmn Ifnar1tm1.2Ees/J |
| 037043 | B6.Cg-Tg(K18-ACE2)2Prlmn Tg(FCGRT)32Dcr Fcgrttm1Dcr/2J |
| 034902 | B6.Cg-Tg(K18-ACE2)2Prlmn Tg(FCGRT)32Dcr Fcgrttm1Dcr/J |
| 034860 | B6.Cg-Tg(K18-ACE2)2Prlmn/J |
| 035247 | C.Cg-Tg(K18-ACE2)2Prlmn/J |
| 034901 | NOD.Cg-Tg(K18-ACE2)2Prlmn Prkdcscid Il2rgtm1Wjl/J |
| 7 strains use this protocol | |