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Notes
This assy cannot distinguish hemi from hom
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= 128 bp
IPC = 74 bp
Fam=Mut
Cy5=IPC
Assay cannot distinguish hemi from hom
Sequence
Tg Sequence: GATTAGATAAAAGTAAAGTGATTAACaGCGCATTAGAGcTGCTTAATGaGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTtGGTGTAGAGCAGCCTACAcTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAgCcATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAaAAGGGGAAAGCTGGCAAGATTTTTTACGcAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCaATGGA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40699 | GGT TTA ACA ACC CGT AAA CTC G | Transgene Forward | A | |||
| 40700 | TGA GTA TGG TGC CTA TCT AAC ATC | Transgene Reverse | A | |||
| 40701 | Fluorophore-1 | CAG CCT ACA CTG TAT TGG CAT G | 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 | |||
| 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 | |
| 40699 | 0.40 uM |
| 40700 | 0.40 uM |
| oIMR1544 | 0.40 uM |
| oIMR3580 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
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 |
|---|---|
| 023917 | B6.Cg-Lin28btm1.1Gqda/J |
| 034533 | B6.FVB-Tg(EmuSR-tTa)83Bop/DwfJ |
| 012433 | B6;C3-Tg(ACTA1-rtTA,tetO-cre)102Monk/J |
| 024851 | B6N.129S6-Sod2tm1(rtTA*M2,tetO)Jsg/J |
| 034802 | FVB/N-Tg(MMTV-tTA)25754Kuw/Mmjax |
| 017393 | FVB;B6-CriptTn(sb-rtTA)2427.SB1Ove/Mmjax |
| 005734 | NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ |
| 006999 | STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J |
| 032910 | STOCK Tg(BEST1-rtTA,tetO-cre)1Yzl/J |
| 024854 | STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J |
| 003272 | STOCK Tg(Cebpb-tTA)5Bjd/J |
| 003273 | STOCK Tg(CMV-rtTA)4Bjd/J |
| 025672 | STOCK Tg(tetO-Fgfr2b/Igh)1.3Jaw/J |
| 026194 | STOCK Tg(Wnt1-rtTA2S*M2)1Whsu/J |
| 14 strains use this protocol | |