Protocol 28182: Standard PCR Assay - Tg(TRAMP)8247Ng
Version 3.2

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

Transgene Tm = 83
Internal positive control Tm = 86

IC= 415 bp
Tg= 264 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10363 TAC AAC TGC CAA CTG GGA TG Transgene Forward A
10364 CAG GCA CTC CTT TCA AGA CC Transgene Reverse A
21238 CTG TCC CTG TAT GCC TCT GG Internal Positive Control Forward A
21239 AGA TGG AGA AAG GAC TAG GCT ACA 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
10363 0.50 uM
10364 0.50 uM
21238 0.50 uM
21239 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
008215 (C57BL/6-Tg(TRAMP)8247Ng/J x FVB/NJ)F1/J
003135 C57BL/6-Tg(TRAMP)8247Ng/J
2 strains use this protocol