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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.
TG= 88 bp
IPC= 74 bp
Tg Sequence:
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 |
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 |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | repeat steps 2-3 for 40 cycles |
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 |