Protocol 20782: Probe Assay - GCaMP6f-Probe
Version 1.0

Notes

This assay cannot distinguish hemi from hom

 This assay can distinguish GCaMP6f from GCaMP6s and GCaMP3

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=  95 bp

IPC = 74 bp

Sequence

tcgcagaatttaaagaggAAttctccctatttgacaaggacggggatggga

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31035 GGA CCA ACT GAC TGA AGA GCA Forward A
31036 CCA GCT CCT TGG TTG TTA TTG Reverse A
31038 Fluorophore-1 CAG AAT TTA AAG AGG AAT TCT CCC TAT 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
31035 0.40 uM
31036 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 4.0 -- 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
024103 B6;129S6-Igs7tm93.1(tetO-GCaMP6f)Hze/J
024339 C57BL/6J-Tg(Thy1-GCaMP6f)GP5.11Dkim/J
028281 C57BL/6J-Tg(Thy1-GCaMP6f)GP5.12Dkim/J
025393 C57BL/6J-Tg(Thy1-GCaMP6f)GP5.17Dkim/J
028279 C57BL/6J-Tg(Thy1-GCaMP6f)GP5.1Dkim/J
028280 C57BL/6J-Tg(Thy1-GCaMP6f)GP5.3Dkim/J
024276 C57BL/6J-Tg(Thy1-GCaMP6f)GP5.5Dkim/J
7 strains use this protocol