Protocol 21088: Probe Assay - Generic Amp Probe
Version 2.0

Notes

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=120bp

IPC=74bp

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31698 ACG CTC GTC GTT TGG TAT G Mutant Forward A
31699 CTG ACA ACG ATC GGA GGA C Mutant Reverse A
31700 Fluorophore-1 TCA AGG CGA GTT ACA TGA TCC Quencher-1 MUT 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
31698 0.40 uM
31699 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
039888 B6(129P2)-Ddx3xem1Tdk/J
037318 B6(Cg)-H3c6em1Sjes/J
037333 B6.Cg-Chodlem1(cre)Ngai/TasicJ
029481 B6.Cg-Pax9em1(cre/ERT2)Ychai/J
034424 B6;129S6-Sncgem1(flpo)Tasic/J
040087 C57BL/6-Chd8em1Fezh/TabbJ
037451 C57BL/6-Ifitm3em1Jsy/J
033120 C57BL/6J-Il2rgem1Lutzy/J
033122 C57BL/6J-Il2rgem3Lutzy/J
033032 C57BL/6J-Rab39bem6Lutzy/Mmjax
034905 C57BL/6J-Trex1em1Lutzy/J
11 strains use this protocol