Stock No: 003274
Protocol 24893: Standard PCR Assay - Luciferase: Tg(tetNZL)2Bjd, Tg(tetL)1Bjd, Tg(Fos-luc)1Rnd
Version 1.2

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 = 465 bp

Internal positive control = 324 bp

 

Transgene   Tm = 82     Area = 11       Tm = 85.5   Area = 30

IPC                 Tm = 82     Area = 50

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0960 TCC TCT GAC ACA TAA TTC GCC Transgene Forward A
oIMR0961 GTT GTT CCA TTC CAT CAC GG Transgene Reverse A
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward B
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Internal Positive Control Reverse B

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR0960 0.50 uM
oIMR0961 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.

Reaction B

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
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
003479 B6.C3-Tg(Fos-luc)1Rnd/J
003275 STOCK Tg(tetL)1Bjd/J
003274 STOCK Tg(tetNZL)2Bjd/J
3 strains use this protocol