Protocol 28487: Standard PCR Assay - Tg(Thy1-GCaMP)
Version 2.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

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

Transgene = 248 bp

Internal positive control = 320 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
18911 CAT CAG TGC AGC AGA GCT TC Transgene Forward A
18912 CAG CGT ATC CAC ATA GCG TA Transgene Reverse A
21157 CTC CCA ACC CCA GAG GTA GT Internal Positive Control Forward A
21225 AGA CCC CAG ATC CAG AAA GG Internal Positive Control Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
18911 0.50 uM
18912 0.50 uM
21157 0.50 uM
21225 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
028277 C57BL/6J-Tg(Thy1-GCaMP3)GP2.11Dkim/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
025776 C57BL/6J-Tg(Thy1-GCaMP6s)GP4.12Dkim/J
024275 C57BL/6J-Tg(Thy1-GCaMP6s)GP4.3Dkim/J
028278 C57BL/6J-Tg(Thy1-GCaMP6s)GP4.6Dkim/J
10 strains use this protocol