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Mutant (28555, 28551) (Rsky) = 172 bp
Mutant (30936, 26179 and 24858) (Fezh, Dvs, & Mvw) = 290 bp
Wild type = 109 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
21306 | CTG GCT TCT GAG GAC CG | Wild type Forward | A | |||
24493 | AGC CTG CCC AGA AGA CTC C | Wild type Reverse | A | |||
26932 | CTC CGT CGT GGT CCT TAT AGT | Mutant Reverse | A | |||
oIMR5984 | GCT AAC CAT GTT CAT GCC TTC | Mutant Forward | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
21306 | 0.50 uM |
24493 | 0.50 uM |
26932 | 0.50 uM |
oIMR5984 | 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 |
---|---|
028555 | B6(C)-Gt(ROSA)26Sorem1.1(CAG-cas9*,-EGFP)Rsky/J |
026179 | B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
026430 | B6N.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
028551 | C57BL/6N-Gt(ROSA)26Sorem1(CAG-cas9*,-EGFP)Rsky/J |
026558 | FVB.129(B6)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
026557 | NOD.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
030936 | NOD/ShiLtJ-Gt(ROSA)26Sorem1.1(CAG-cas9*,-EGFP)Dvs/J |
024858 | STOCK Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J |
8 strains use this protocol |