Protocol 28963: Standard PCR Assay - Tg(Prnp-ITM2B/APP695*40)1Emcg
Version 2.3

Notes

Melting curve analysis is done using a Roche Light Cycler 480.

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

TG = 307 bp


internal control = 420 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0647 CAG ACA TCG AGG AAG TGA AGG Wild type A
oIMR0648 TGC TGT GGT CAT CAG GTA GG Wild type A
oIMR7042 AAG GCT GGA ACC TAT TTG CC Transgene Forward A
oIMR7043 CGT TAC TAG TGG ATC CCT AG 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
oIMR0647 0.50 uM
oIMR0648 0.50 uM
oIMR7042 0.50 uM
oIMR7043 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

This is the only strain that uses this protocol.