Stock No: 030386
Protocol 27653: Standard PCR Assay - Generic SV40 TAg 1
Version 3.2

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

Internal Control = ~320 bp
TG = ~500 bp

 

WT = 87.5

Tg = 83.5

Separate digested products on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21157 CTC CCA ACC CCA GAG GTA GT Internal Positive Control Forward A
21225 AGA CCC CAG ATC CAG AAA GG Internal Positive Control Reverse A
oIMR0068 CAG AGC AGA ATT GTG GAG TGG Transgene A
oIMR0069 GGA CAA ACC ACA ACT AGA ATG CAG TG Transgene A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
21157 0.50 uM
21225 0.50 uM
oIMR0068 0.50 uM
oIMR0069 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
003446 C57BL/6J-Tg(Amy1TAg)501Knw/J
013591 FVB-Tg(C3-1-TAg)cJeg/JegJ
030386 FVB/N-Tg(C3-1-TAg)cJeg/2JegJ
3 strains use this protocol