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Mutant = 121 bp
Heterozygote = 121 bp and 404 bp
Wild type = 404 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
11910 | GAG ATG TCC CTG AAC CCA GA | Wild type Forward | A | |||
11911 | TTG AGT GAG GAT TGC ACT GG | Wild type Reverse | A | |||
25578 | CGG TCC TAA GGT AGC GAC T | Mutant Forward | B | vector | ||
25579 | TGT TTA TGA TTG AAA GTA GAT TCA GAG | Mutant Reverse | B | Rag2 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
11910 | 0.50 uM |
11911 | 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 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
25578 | 0.50 uM |
25579 | 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 |
---|---|
017712 | C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv Tg(SIRPA)1Flv/J |
017711 | C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J |
017708 | C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Il2rgtm1.1Flv/J |
014595 | C;129S4-Rag2tm1.1Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Il2rgtm1.1Flv/J |
017707 | C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv Tg(SIRPA)1Flv/J |
014593 | C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J |
014594 | C;129S4-Rag2tm1.1Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J |
7 strains use this protocol |