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Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
9414 | TTC AGA CAC TGC GTG ACT | Mutant Forward | A | birA | ||
9415 | GGC TCC AAT GAC TAT TTG C | Mutant Reverse | A | birA | ||
9416 | GTG TAA CTG TGG ACA GAG GAG | Wild type Forward | B | |||
9417 | GAA CTT GAT GTG TAG ACC AGG | Wild type Reverse | B |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
9414 | 0.50 uM |
9415 | 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 |
9416 | 0.50 uM |
9417 | 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 |
---|---|
010920 | FVB;129P2-Gt(ROSA)26Sortm1.1(birA)Mejr/J |
018121 | STOCK Gt(ROSA)26Sortm1(birA)Mejr Gata4tm3.1Wtp/J |
025980 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Ep300tm1.1Wtp/Mmjax |
025983 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Mef2ctm1.1Wtp/Mmjax |
025978 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Nkx2-5tm1.1Wtp/Mmjax |
025979 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Srftm1.1Wtp/Mmjax |
025981 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Tead1tm1.1Wtp/Mmjax |
025977 | STOCK Gt(ROSA)26Sortm1.1(birA)Mejr Zfpm2tm1.1Wtp /Mmjax |
025982 | STOCK Rbpjtm1.1Wtp Gt(ROSA)26Sortm1.1(birA)Mejr/Mmjax |
9 strains use this protocol |