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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).
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
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 |
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 |
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 |
Stock Number | Strain Name |
---|---|
017700 | B6.129-Rag1tm1Mom Fcgrttm1Dcr/DcrJ |
002216 | B6.129S7-Rag1tm1Mom/J |
016919 | B6.Cg-Rag1tm1Mom Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ |
004369 | B6.Cg-Rag1tm1Mom Ins2Akita/J |
008684 | B6.Cg-Rag1tm1Mom Tyrp1B-w Tg(Tcra,Tcrb)9Rest/J |
013115 | B6.Cg-Rag1tm1Mom Tg(UBC-GFP)30Scha/J |
002096 | B6;129S7-Rag1tm1Mom/J |
021571 | BXSB.129S7(B6)-Rag1tm1Mom/DcrJ |
003145 | C.129S7(B6)-Rag1tm1Mom/J |
030666 | C.Cg-Rag1tm1Mom Tg(DO11.10)10Dlo/J |
003729 | NOD.129S7(B6)-Rag1tm1Mom/J |
004347 | NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ |
004348 | NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ |
030442 | NOD.Cg-Rag1tm1Mom Dmdmdx-4Cv Il2rgtm1Wjl/McalJ |
026062 | NOD.Cg-Rag1tm1Mom Fancaem1Dvs Il2rgtm1Wjl/SzJ |
006022 | NOD.Cg-Rag1tm1Mom H2-Ab1b-tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ |
006024 | NOD.Cg-Rag1tm1Mom H2-Ab1b-tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ |
008659 | NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ |
026014 | NOD.Cg-Rag1tm1Mom KitW-41J Il2rgtm1Wjl/EavJ |
004848 | NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/SzJ |
030443 | NOD.Cg-Rag1tm1Mom Sgcatm1Kcam Il2rgtm1Wjl/McalJ |
029663 | NOD.Cg-Rag1tm1Mom Dysfprmd Il2rgtm1Wjl/McalJ |
024099 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/J |
017914 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/ScasJ |
007799 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ |
014568 | NOD.Cg-Rag1tm1Mom Ins2Akita Il2rgtm1Wjl/SzJ |
009377 | NOD.Cg-Rag1tm1Mom Tg(TcraBDC12-4.1)10Jos Tg(TcrbBDC12-4.1)82Gse/J |
005707 | STOCK Rag1tm1Mom Tg(TIE2-lacZ)182Sato/J |
006770 | STOCK Rag1tm1Mom Tg(TIE2GFP)287Sato/J |
29 strains use this protocol |