Stock No: 034711
Protocol 18722: Standard PCR Assay - Generic Hygromycin Alternate1
Version 2.0

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

 Tg  = ~210 bp
IPC = 320 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21157 CTC CCA ACC CCA GAG GTA GT Internal Positive Control Forward A
21225 AGA CCC CAG ATC CAG AAA GG Internal Positive Control Reverse A
oIMR1020 CGG AAG TGC TTG ACA TTG G Transgene A
oIMR1021 GTA TTG ACC GAT TCC TTG CG Transgene A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
21157 0.50 uM
21225 0.50 uM
oIMR1020 0.50 uM
oIMR1021 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
030985 B6.129P2(Cg)-Aph1atm1Bdes Aph1btm1Bdes Aph1ctm1Bdes/J
034711 B6.Cg-Apptm1.1Dnli/J
029604 B6.Cg-Col1a1tm1(CAG-Sirt2)Jmi/DsinJ
034797 B6.Cg-Gt(ROSA)26Sortm2(CAG-rtTA3,-mKate2)Slowe Col1a1tm1(tetO-GFP,-PRKAA1*)Rshaw/J
035404 B6;129S-Igs7tm166(tetO-EGFP*,CAG-tTA2)Hze/XwyJ
034422 B6;129S6-Slc17a7em1(flpo)Hze/J
014594 C;129S4-Rag2tm1.1Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J
029931 C57BL/6-Firretm1Rinn/J
026872 C57BL/6-Hamptm1Ddhf/J
003127 FVB;129P2-Bcl3tm1Ver/J
10 strains use this protocol