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E4= GC/C = Mut JR 27894/28709
E2= CT/T = Mut JR 29017
E3= CT/C = JR 29018
B6 wt mouse will have no PCR product
>chr19:44908623+44908846 224bp ACGGCTGTCCAAGGAGCTG CCCCGGCCTGGTACACTG
ACGGCTGTCCAAGGAGCTGCAGGCGGCGCAGGCCCGGCTGGGCGCGGACATGGAGGACGT(gc/ct)GCGGCCGCCTGGTGCAGTACCGCGG
CGAGGTGCAGGCCATGCTCGGCCAGAGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCC
CACCTGCGCAAGCTGCGTAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAG(c/t)GC
CTGGCAGTGTACCAGGCCGGGGCCC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
24459 | CCC CGG CCT GGT ACA CTG | Reverse | A | |||
29961 | ACG GCT GTC CAA GGA GCT G | Forward | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
24459 | 0.50 uM |
29961 | 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 |