Protocol 20345: Standard PCR Assay - Generic rtTA-Alternate 1
Version 5.0

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

TG = ~300 bp
Hemizygotes = ~300 bp and 415 bp
IPC = 415 bp

Assay cannot distinguish hemi from hom

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
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
oIMR8747 CAT GTC CAG ATC GAA ATC GTC Transgene Reverse A rtTA
oIMR8932 CTG TTC GGC CTT GAA TTG AT Transgene Forward A rtTA

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
21238 0.50 uM
21239 0.50 uM
oIMR8747 0.50 uM
oIMR8932 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
023917 B6.Cg-Lin28btm1.1Gqda/J
034533 B6.FVB-Tg(EmuSR-tTa)83Bop/DwfJ
012433 B6;C3-Tg(ACTA1-rtTA,tetO-cre)102Monk/J
024851 B6N.129S6-Sod2tm1(rtTA*M2,tetO)Jsg/J
034802 FVB/N-Tg(MMTV-tTA)25754Kuw/Mmjax
005734 NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ
006999 STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J
024854 STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J
003272 STOCK Tg(Cebpb-tTA)5Bjd/J
003273 STOCK Tg(CMV-rtTA)4Bjd/J
025672 STOCK Tg(tetO-Fgfr2b/Igh)1.3Jaw/J
026194 STOCK Tg(Wnt1-rtTA2S*M2)1Whsu/J
12 strains use this protocol