Genotyping Protocols Database
Protocol 22455: Standard PCR Assay - Fcgrt<tm1Dcr>
Version 1.3

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

Mutant = 378 bp
Heterozygote = 248 bp and 378 bp
Wild type = 248 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
oIMR7720 GGG ATG CCA CTG CCC TG Wild type Forward A
oIMR7721 CGA GCC TGA GAT TGT CAA GTG TAT T Common A
oIMR7722 GGA ATT CCC AGT GAA GGG C Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR7720 0.50 uM
oIMR7721 0.50 uM
oIMR7722 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
017700 B6.129-Rag1tm1Mom Fcgrttm1Dcr/DcrJ
003982 B6.129X1-Fcgrttm1Dcr/DcrJ
021146 B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(CAG-FCGRT)276Dcr/DcrJ
018441 B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(FCGRT)32Dcr/DcrJ
018541 B6.Cg-Fcgrttm1Dcr Prkdcscid/DcrJ
004919 B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ
014565 B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ
016919 B6.Cg-Rag1tm1Mom Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ
025201 B6.Cg-Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr/MvwJ
021455 BXSB.129X1-Fcgrttm1Dcr/DcrJ
003986 MRL.129X1-Fcgrttm1Dcr/DcrJ
003988 NOD.129X1-Fcgrttm1Dcr/DvsJ
003989 NZM2410.129X1-Fcgrttm1Dcr/DcrJ
003990 SJL.129X1-Fcgrttm1Dcr/DcrJ
14 strains use this protocol