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>chr1:71065419-71065753 335bp GACCGACTTGAGTTCCCACT CTGCTTCGGTACACATCACC
Mut = C
WT= G
The forward primer (primer 61373) anneals over the nucleotide sequence containing mouse genomic variation rs32907285 and the reverse primer (primer 61374) anneals over the nucleotide sequence containing mouse genomic variation rs32907287.
gaccgacttgagttcccactactttctctaatgtgtatttattaacacactgttttcccatcttcttcccacttgccctatttgaaactttgtttggattagCATgAAGCCTGCAGTCATGGGCACCTGAAGGTAGTGGAGTTGCTGCTCCAGCATAATGCCCTGGTGAACACCCCTGGCTATCAGAATGACTCGCCACTCCAC(g/c)ATGCCGTCAAGAGTGGCCACATCGATATAGTCAAGGTGTTACTGTCCCACGGTGCTTCCAGGAATGCTGTgtaagtatctgattttcaaaatggatttgtgttcctgaatggtgatgtgtaccgaagcag
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
61373 | GAC CGA CTT GAG TTC CCA CT | Forward | A | |||
61374 | CTG CTT CGG TAC ACA TCA CC | Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
61373 | 0.50 uM |
61374 | 0.50 uM |
Glycerol | 6.50 % |
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 |