Protocol 18180: Separated PCR Assay - Tg(FCGRT) alternate1
Version 5.0

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

JR4915, huFcRn line 32, will give a transgene band ~740 bp. 
JR4919, huFcRn line 276 will give a product ~350 bp (because 32 is based on genomic DNA and 276 on cDNA).

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
oIMR7718 AGC CAA GTC CTC CGT GCT C Transgene Forward A FCGRT
oIMR7719 CTC AGA GAT GCC AGT GTT CC Transgene Reverse A FCGRT
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward B
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control Reverse B

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR7718 0.50 uM
oIMR7719 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
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
021146 B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(CAG-FCGRT)276Dcr/DcrJ
004919 B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ
016919 B6.Cg-Rag1tm1Mom Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ
029686 B6.Cg-Tg(FCGRT)32Dcr Fcgrttm1Dcr Ighg1em2(IGHG1)Mvw/MvwJ
025099 BXSB.Cg-Fcgrttm1Dcr Tg(FCGRT)99Dcr/J
028616 NOD.Cg-Tg(CAG-FCGRT)276Dcr Fcgrttm1Dcr Prkdcscid Il2rgtm1Wjl/J
6 strains use this protocol