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Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler 480. 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.
Archived Notes:
Name of protoocl changed from Tg(Il2/NFAT-luc)83Rinc to generic Luc qPCR 12-13-17 ESP
Removed 13745 since it had a Fam label and replaced with 105 12-18-17 ESP
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13736 | Fluorophore-1 | TGG GCG CGT TAT TTA TCG GAG TTG C | Quencher-1 | Tg Probe | ||
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 | |||
oIMR6615 | CTC TTC AAT TCT TTA TGC CGG TG | Transgene Forward | A | |||
oIMR6616 | TTC GCG GGC GCA AC | Transgene Reverse | A | |||
TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa Probe Fast QPCR | 1.00 X |
oIMR1544 | 0.40 uM |
oIMR3580 | 0.40 uM |
oIMR6615 | 0.40 uM |
oIMR6616 | 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 |
---|---|
033605 | B6.Cg-S1pr1tm4.1Rlp Gt(ROSA)26Sortm2.1(luc*/Arrb2*)Rlp Tyrc-2J/J |
006098 | B6.Cg-Tg(Il2/NFAT-luc)83Rinc/J |
006851 | B6.Cg-Tg(Per1-luc)025Jt/J |
027662 | C57BL/6-Tyrc-Brd Tg(Gh1-luc/EGFP)D8Mrln/J |
007800 | FVB/N-Tg(Ins1-luc)VUPwrs/J |
012370 | FVB/NJ-Tg(Hspa1a-luc,-EGFP)2Chco/J |
017597 | STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4299Ahmb Tg(tetO-SMN2,-luc)#bAhmb/J |
7 strains use this protocol |