Stock No: 007800
Protocol 28551: Standard PCR Assay - Generic Luc
Version 5.2

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 = 572 bp
Internal Positive Control = 200 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9767 GAA ATG TCC GTT CGG TTG GCA GAA GC Transgene Forward A Luc
9768 CCA AAA CCG TGA TGG AAT GGA ACA ACA Transgene Reverse A Luc
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT 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
9767 0.50 uM
9768 0.50 uM
oIMR8744 0.50 uM
oIMR8745 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
006100 B10.Cg-H2k Tg(NFkB/Fos-luc)26Rinc/J
033605 B6.Cg-S1pr1tm4.1Rlp Gt(ROSA)26Sortm2.1(luc*/Arrb2*)Rlp Tyrc-2J/J
023429 B6.Cg-Tyrc-2J Cdkn1atm1Hpw/J
006098 B6.Cg-Tg(Il2/NFAT-luc)83Rinc/J
006101 B6.Cg-Tg(TRE/Prl-luc)31FlvRinc/J
002709 B6;C3-Tg(TettTALuc)1Dgs/J
007800 FVB/N-Tg(Ins1-luc)VUPwrs/J
032442 FVB/N-Tg(tetO/CMV-Dek,-luc)317Siwe/J
012370 FVB/NJ-Tg(Hspa1a-luc,-EGFP)2Chco/J
9 strains use this protocol