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JR 24418
Mut = GAT and GAC
WT= TCC and AGC
JR 24420
Mut = GCAGCAGCC
wt = ACATCGACC
JR 24418
GGTTTCTGTTTGATTCCACAGCAA(TCC/GAT)atcaatgaagccatacgg
acatcgaccctccccaggaac(AGC/GAC)GGGGCAGGAGCCAGCGGAGG
AAGTGGCAGTGGAGAGAATGG
JR 24420
ccATCAATGAAGCCATACGGa/gcat/gcga/agccCTC
CCCAGGAAC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
20862 | TTG GGC AAA AGT TTC AAT CC | Forward | A | |||
20863 | GAA GTC CTG GCT GAC CAC TC | Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
20862 | 0.50 uM |
20863 | 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 |