Protocol 28254: Standard PCR Assay - Generic tTA
Version 2.2

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 = 92°C +/- 1.0°C
Internal positive control Tm = 82°C+/- 1.0°C

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control Reverse A
oIMR8746 CGC TGT GGG GCA TTT TAC TTT AG Transgene Forward A
oIMR8747 CAT GTC CAG ATC GAA ATC GTC 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
oIMR8744 0.50 uM
oIMR8745 0.50 uM
oIMR8746 0.50 uM
oIMR8747 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
016176 B6(Cg)-Tg(tetO-Per2)2Jt/J
028295 B6.Cg-Tg(tetO-DRD3)3-10Kndl/J
017719 C3HeB/FeJ-Tg(tetO-TAg)1Efr/J
034802 FVB/N-Tg(MMTV-tTA)25754Kuw/Mmjax
004602 NOD.Cg-Tg(Ins2-rtTA)2Doi/DoiJ
006999 STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J
018999 STOCK Gt(ROSA)26Sortm1(tTA,tetO-Mir155)Fjsl/J
024854 STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J
025672 STOCK Tg(tetO-Fgfr2b/Igh)1.3Jaw/J
9 strains use this protocol