Protocol 29404: Standard PCR Assay - Firefly Luc
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 = 200 bp
Internal positive control = 324 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10946 AAA TCG CTC CGG ATA CTG CG Transgene Forward A firefly luc
10947 AGG GTT GGT ACT AGC AAC GC Transgene Reverse A firefly luc
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10946 0.50 uM
10947 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
006100 B10.Cg-H2k Tg(NFkB/Fos-luc)26Rinc/J
025854 B6;FVB-Ptprca Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J
010545 C.FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ
027832 FVB/N-Tg(ABCB1-luc)1Egsch/J
025855 STOCK Ptprca Lag3tm1Doi Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J
5 strains use this protocol