Stock No: 003185
Protocol 26590: Standard PCR Assay - Tg(PRG)
Version 3.2

Notes

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

TG TM = 90.5°C
Internal Control TM = 88.5°C 

TG = 226 bp
Internal Control = 155 bp
 

Sequence

TG TM = 90.5°C
Internal Control TM = 88.5°C
 
TG = 226 bp
Internal Control = 155 bp
 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0180 GCC TAG CCG AGG GAG AGC CG Wild type A
oIMR0181 TGT GAC TTG GGA GCT CTG CAG C Wild type A
oIMR0225 CCA CCC AGG TGC CCG GCT TCT T Transgene A
oIMR0226 CTC AGC GAG GCA TTC CGC AGC Transgene A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0180 0.50 uM
oIMR0181 0.50 uM
oIMR0225 0.50 uM
oIMR0226 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
003185 C57BL/6-Tg(PRG1)18Wlad/J
003186 C57BL/6-Tg(PRG3)9Wlad/J
2 strains use this protocol