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Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR1750 | CCG TCT AGC TTG AGC TAT TAG G | Common | A | |||
oIMR1751 | GAA GAG GAC GAT GAA GGT GG | Wild type Reverse | A | |||
oIMR5255 | TTG TGC CCA GTC ATA GCC GAA T | Mutant Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
oIMR1750 | 0.50 uM |
oIMR1751 | 0.50 uM |
oIMR5255 | 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 |
---|---|
002249 | B10.129S2(B6)-Ighmtm1Cgn/J |
002288 | B6.129S2-Ighmtm1Cgn/J |
021871 | BXSB.129S2(B6)-Ighmtm1Cgn/DcrJ |
004349 | C.129S2-Ighmtm1Cgn/J |
004639 | NOD.129S2(B6)-Ighmtm1Cgn/DoiJ |
003903 | NOD.129S2(B6)-Ighmtm1Cgn/Dvs |
006608 | NOD.Cg-Ighmtm1Cgn Tg(IghelMD4)4Ccg/DvsJ |
005309 | NODCaj.Cg-Ighmtm1Cgn Tg(Igh-VB1-8/Igh-6)2Mjsk/FswJ |
005306 | NODCaj.Cg-Ighmtm1Cgn Tg(Igh-VB1-8/Igh-6m)1Mjsk/FswJ |
9 strains use this protocol |