Protocol 28768: Standard PCR Assay - Generic DsRed
Version 6.2

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

Transgene Tm = 87.9°C +/- 1.0°C
Internal positive control Tm = 82.7°C+/- 1.0°C

Tg = 217 bp

IPC = 324 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR6787 CCC ATG GTC TTC TTC TGC AT Transgene Forward A
oIMR6788 AAG GTG TAC GTG AAG CAC CC 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
oIMR6787 0.50 uM
oIMR6788 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
006055 B6.Cg-Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J
018410 B6.Cg-Tg(Pnkd*A7V*A9V,-DsRed)671Ljp/J
013082 B6.FVB-Tg(Per2-DsRed*T3)12Obr/Mmjax
006679 B6;129P2-Olfr151tm27Mom/MomJ
008605 B6;C3-Tg(CAG-DsRed,-EGFP)5Gae/J
009655 C57BL/6J-Tg(Dcx-DsRed)14Qlu/J
018068 FVB-Tg(Prism)1989Htz/J
017620 NOD.Cg-Prkdcscid Tg(CAG-DsRed*MST)1Nagy/KupwJ
005328 NOD/ShiLt-Tg(Cd4-DsRed)4Lt/J
005441 STOCK Tg(CAG-DsRed*MST)1Nagy/J
006866 STOCK Tg(Ins1-DsRed*T4)32Hara/J
11 strains use this protocol