Protocol 24371: Separated PCR Assay - Iis (GT vs TG)
Version 1.2

Notes

For Iis (GT vs TG) the assay is used to tell the orientation of the mutation it does not have a band for WT. Thus SIQ = lack of mutant band.

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

GT - ~300 bp

TG- ~326 bp

Use specific assays for WT.

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
17242 CGC ACC ATC TTC TTC AAG G Mutant Forward A
17243 CCA AGC TGA AGG TGA CCA AG Mutant Forward B
17244 TCT TCT TCT GCA TTA CGG GG Mutant Reverse B
oIMR3999 TGC TCA GGT AGT GGT TGT CG Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
17242 0.50 uM
oIMR3999 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.

Reaction B

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
17243 0.50 uM
17244 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
021458 STOCK Iis5tm1(ACTB-tdTomato,-EGFP)Luo/J
021457 STOCK Iis5tm2.1(ACTB-EGFP,-tdTomato)Luo/J
021461 STOCK Iis6tm1.1(ACTB-tdTomato,-EFGP)Luo/J
021460 STOCK Iis6tm2.1(ACTB-EFGP,-tdTomato)Luo/J
4 strains use this protocol