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Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
9655 | CCA GGC GGG CCA TTT ACC GTA AG | Mutant | A | CAG | ||
oIMR8545 | AAA GTC GCT CTG AGT TGT TAT | Common | A | |||
oIMR8546 | GGA GCG GGA GAA ATG GAT ATG | Wild type | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
9655 | 0.50 uM |
oIMR8545 | 0.50 uM |
oIMR8546 | 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 |
---|---|
024846 | B6;129S-Gt(ROSA)26Sortm2.1Ksvo/J |
023035 | B6;129S6-Gt(ROSA)26Sortm1(CAG-tdTomato*,-EGFP*)Ees/J |
023537 | B6N.129S6-Gt(ROSA)26Sortm1(CAG-tdTomato*,-EGFP*)Ees/J |
010675 | STOCK Gt(ROSA)26Sortm1(CAG-EGFP)Fsh/Mmjax |
010701 | STOCK Gt(ROSA)26Sortm1.1(CAG-EGFP)Fsh/Mmjax |
010812 | STOCK Gt(ROSA)26Sortm1.2(CAG-EGFP)Fsh/Mmjax |
6 strains use this protocol |