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Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR1241 | ATG ACC GAG TAC AAG CCC AC | Mutant Forward | A | Puro F | ||
oIMR1242 | GCG TGA GGA AGA GTT CTT GC | Mutant Reverse | A | Puro R | ||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
oIMR1241 | 0.50 uM |
oIMR1242 | 0.50 uM |
oIMR7338 | 0.50 uM |
oIMR7339 | 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 |
---|---|
024906 | 129-Dp(16Tiam1-Il10rb)8Yey/J |
035506 | B6.Cg-Gt(ROSA)26Sortm3(CEBPB)Myz/J |
014168 | B6;129-Pot1atm1.1Tdl/J |
038621 | B6;129P2-Ctcftm2.2Gal/J |
002355 | C57BL/6J-Tg(pPWL88puro)2Ems/J |
003208 | STOCK Dnmt1tm3Jae Hprt1b-m3 Tg(pPWL512hyg)1Ems/J |
018121 | STOCK Gt(ROSA)26Sortm1(birA)Mejr Gata4tm3.1Wtp/J |
028980 | STOCK Ppm1dtm1Lad/J |
8 strains use this protocol |