Genotyping Protocols Database
Protocol 18439: Standard PCR Assay - Tg(Camk2a-tTA)1Mmay
Version 1.0

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 = ~550 bp

Internal positive control = 324 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25348 GAC CTG GAT GCT GAC GAA GG Transgene Forward A Camk2a
27058 GCA GCT CTA ATG CGC TGT Transgene Reverse A tTA
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C 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
25348 0.50 uM
27058 0.50 uM
oIMR7338 0.50 uM
oIMR7339 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
003010 B6;CBA-Tg(Camk2a-tTA)1Mmay/J
007004 B6.Cg-Tg(Camk2a-tTA)1Mmay/DboJ
016198 129S6.Cg-Tg(Camk2a-tTA)1Mmay/JlwsJ
024108 STOCK Igs7tm93.1(tetO-GCaMP6f)Hze Tg(Camk2a-tTA)1Mmay/J
024115 B6.Cg-Igs7tm94.1(tetO-GCaMP6s)Hze Tg(Camk2a-tTA)1Mmay/J
024854 STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J
025105 FVB.Cg-Tg(Camk2a-tTA)1Mmay/DboJ
7 strains use this protocol