Genotyping Protocols Database
Protocol 27761: Standard PCR Assay - Rag1<tm1Mom>Alternate1
Version 1.2

Notes

To run this assay on agarose you will need to run two separate reactions; one for the wild type Rag1 allele (Wild type forward and common primers) and one for the Rag1 knockout allele (mutant forward and common primers).

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 = 197 bp
Heterozygote = 197 bp and 192 bp
Wild type = 192 bp

If running on a gel run separated:

23267and 23268 for Wt

23268 and oIMR8162 for Mut

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
23267 TCT GGA CTT GCC TCC TCT GT Wild type Forward A
23268 CAT TCC ATC GCA AGA CTC CT Common A
oIMR8162 TGG ATG TGG AAT GTG TGC GAG Mutant Forward A PGK R

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
23267 0.50 uM
23268 0.50 uM
oIMR8162 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
002096 B6;129S7-Rag1tm1Mom/J
002216 B6.129S7-Rag1tm1Mom/J
003145 C.129S7(B6)-Rag1tm1Mom/J
003729 NOD.129S7(B6)-Rag1tm1Mom/J
004347 NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ
004348 NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ
004369 B6.Cg-Rag1tm1Mom Ins2Akita/J
004848 NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/SzJ
005707 STOCK Rag1tm1Mom Tg(TIE2-lacZ)182Sato/J
006022 NOD.Cg-Rag1tm1Mom H2-Ab1tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ
006024 NOD.Cg-Rag1tm1Mom H2-Ab1tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ
006770 STOCK Rag1tm1Mom Tg(TIE2GFP)287Sato/J
007799 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ
008659 NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ
008684 B6.Cg-Rag1tm1Mom Tyrp1B-w Tg(Tcra,Tcrb)9Rest/J
009377 NOD.Cg-Rag1tm1Mom Tg(TcraBDC12-4.1)10Jos Tg(TcrbBDC12-4.1)82Gse/J
013115 B6.Cg-Rag1tm1Mom Tg(UBC-GFP)30Scha/J
014568 NOD.Cg-Rag1tm1Mom Ins2Akita Il2rgtm1Wjl/SzJ
016919 B6.Cg-Rag1tm1Mom Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ
017700 B6.129-Rag1tm1Mom Fcgrttm1Dcr/DcrJ
017914 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/ScasJ
021571 BXSB.129S7(B6)-Rag1tm1Mom/DcrJ
024099 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/J
026014 NOD.Cg-Rag1tm1Mom KitW-41J Il2rgtm1Wjl/EavJ
026062 NOD.Cg-Rag1tm1Mom Fancaem1Dvs Il2rgtm1Wjl/SzJ
029663 NOD.Cg-Rag1tm1Mom Dysfprmd Il2rgtm1Wjl/McalJ
030442 NOD.Cg-Rag1tm1Mom Dmdmdx-4Cv Il2rgtm1Wjl/McalJ
030443 NOD.Cg-Rag1tm1Mom Sgcatm1Kcam Il2rgtm1Wjl/McalJ
030666 C.Cg-Rag1tm1Mom Tg(DO11.10)10Dlo/J
29 strains use this protocol