Protocol 27228: Standard PCR Assay - Tg(Foxp3-DTR/EGFP)23.2Spar
Version 3.2

Notes

This assay does not work well without the use of a Hotstart Taq polymerase.
(a) We want to keep this as a generic statement, so that it covers the multiple
Hotstart Taqs we currently use, and if we change vendors in the future.

This assay will not distinquish 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 = 370 bp
Internal Positive Control = 200 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10012 CCC AGG TTA CCA TGG AGA GA Transgene Forward A
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control Reverse A
oIMR9402 GAA CTT CAG GGT CAG CTT GC Transgene Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10012 0.50 uM
oIMR8744 0.50 uM
oIMR8745 0.50 uM
oIMR9402 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
011010 C.B6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax
011003 C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax
2 strains use this protocol