Stock No: 002599
Protocol 22240: Standard PCR Assay - Tg(KLK4mHEL)6Ccg, Tg(ML5sHEL)5Ccg
Version 1.3

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 = 160 bp
internal control = 324 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
oIMR0327 GAG CGT GAA CTG CGC GAA GA Transgene A
oIMR0328 TCG GTA CCC TTG CAG CGG TT Transgene 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
oIMR0327 0.50 uM
oIMR0328 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
002598 C57BL/6-Tg(KLK4mHEL)6Ccg/J
002599 C57BL/6-Tg(ML5sHEL)5Ccg/J
004258 NOD.B6-Tg(KLK4mHEL)6Ccg/Dvs
004252 NOD.B6-Tg(ML5sHEL)5Ccg/Dvs
006610 NOD.B6-Tg(ML5sHEL)5Ccg/DvsJ
5 strains use this protocol