Genotyping Protocols Database
Protocol 26962: Standard PCR Assay - Tg(tTA)
Version 5.2

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 = 450 bp

Internal positive control = 297 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8746 CGC TGT GGG GCA TTT TAC TTT AG Transgene Forward A
oIMR8747 CAT GTC CAG ATC GAA ATC GTC Transgene Reverse A
oIMR9020 AAG GGA GCT GCA GTG GAG TA Wild type Forward A
oIMR9021 CCG AAA ATC TGT GGG AAG TC Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR8746 0.50 uM
oIMR8747 0.50 uM
oIMR9020 0.50 uM
oIMR9021 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
016198 129S6.Cg-Tg(Camk2a-tTA)1Mmay/JlwsJ
003563 B6.Cg-Tg(Cebpb-tTA)5Bjd/J
003767 B6.Cg-Tg(Eno2tTA)5021Nes/J
003763 B6.Cg-Tg(Eno2tTA)5030Nes/J
005964 B6.Cg-Tg(GFAP-tTA)110Pop/J
002618 B6.Cg-Tg(MMTVtTA)1Mam/J
006232 B6.Cg-Tg(Scgb1a1-rtTA)1Jaw/J
006235 B6.Cg-Tg(SFTPC-rtTA)5Jaw/J
012433 B6;C3-Tg(ACTA1-rtTA,tetO-cre)102Monk/J
007678 B6;SJL-Tg(KRT14-rtTA)208Jek/J
006242 C.Cg-Tg(Scgb1a1-rtTA)1Jaw/J
006245 C.Cg-Tg(SFTPC-rtTA)5Jaw/J
013585 FVB-Tg(Cdh5-tTA)D5Lbjn/J
005625 FVB-Tg(Pcp2-tTA)3Horr/J
006222 FVB.Cg-Tg(Scgb1a1-rtTA)1Jaw/J
006225 FVB.Cg-Tg(SFTPC-rtTA)5Jaw/J
006209 FVB.Cg-Tg(Tal1-tTA)19Dgt/J
004127 FVB/N-Tg(Nes-rtTA)306Rvs/J
005942 FVB/N-Tg(Pf4-tTA/VP16)42Kra/J
006875 FVB/N-Tg(Tagln-rtTA)E1Jwst/J
004937 NOD.Cg-Tg(Ins2-tTA)1Doi/DoiJ
005734 NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ
006999 STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J
003272 STOCK Tg(Cebpb-tTA)5Bjd/J
003273 STOCK Tg(CMV-rtTA)4Bjd/J
003271 STOCK Tg(CMV-tTA)3Bjd/J
008250 STOCK Tg(Ins2-rtTA)2Efr/J
005493 STOCK Tg(Tek-rtTA,TRE-lacZ)1425Tpr/J
008790 STOCK Tg(tetO-DISC1*)1001Plet/J
29 strains use this protocol