Protocol 27978 - Tg(GAPDH-luc)

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 = 551 bp
Internal Positive Control = 324 bp

1.5% gel

JAX Protocol

Protocol Primers

Primer	Sequence 5' → 3'	Primer Type	Reaction
9018	CTC CCA TCG GGC CAA TCT CAG TCC	Transgene Forward	A
9019	GCG TTT GCG TTG CTC GGG GTC GTA	Transgene Reverse	A
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
9018	0.50 uM
9019	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

This is the only strain that uses this protocol.