Genotyping Protocols Database
Stock No: 013144
Protocol 22341: Standard PCR Assay - Generic LacZ
Version 1.3

Notes

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 = 315 bp
Internal positive control = 210 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0040 CGT GGC CTG ATT CAT TCC Transgene Reverse A LacZ R
oIMR3054 ATC CTC TGC ATG GTC AGG TC Transgene Forward A LacZ F
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

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0040 0.50 uM
oIMR3054 0.50 uM
oIMR8744 0.50 uM
oIMR8745 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
002621 B6;SJL-Tg(tetop-lacZ)2Mam/J
002856 FVB/N-Tg(TIE2-lacZ)182Sato/J
002982 B6.Cg-Tg(CAG-lacZ)32And/J
003299 B6;SWJ-Tg(TIMP3-lacZ)7Jeb/J
003534 STOCK Inpp5dtm1Dmt/J
004141 B6;CBA-Tg(UAS-lacZ)65Rth/J
004478 B6.129-Foxd1tm1Lai/J
004623 STOCK Tg(TCF/Lef1-lacZ)34Efu/J
005317 B6.Cg-Tg(BAT-lacZ)3Picc/J
005493 STOCK Tg(Tek-rtTA,TRE-lacZ)1425Tpr/J
005707 STOCK Rag1tm1Mom Tg(TIE2-lacZ)182Sato/J
005728 STOCK Tg(tetO-Ipf1,lacZ)958.1Macd/J
005814 B6.129P2-Grm1tm1Dgen/J
006229 B6.Cg-Tg(DRE-lacZ)2Gswz/J
006465 B6;CBA-Tg(CAG-lacZ-WGA)330Bbm/J
006477 B6.Cg-Tg(CAG-lacZ-WGA)330Bbm/J
006578 STOCK Myoz2tm1Eno/J
006613 STOCK Tg(CAG-Bgeo,-Tle1,-ALPP)1Lbe/J
006665 B6;129P2-Olfr151tm13(Olr226)Mom/MomJ
006672 B6;CBA-Tg(Olfr16*,taulacZ)7Mom/MomJ
006686 STOCK Olfr6tm1Mom/MomJ
008344 B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J
008477 STOCK Tg(RARE-Hspa1b/lacZ)12Jrt/J
012335 STOCK Hprttm50(Ple55-lacZ)Ems/Mmjax
012353 STOCK Hprttm65(Ple53-lacZ)Ems/Mmjax
013144 B6.129P2-Aim2Gt(CSG445)Byg/J
018627 B6;SJL-Tg(Myl1-lacZ)1Ibdml/J
27 strains use this protocol