Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:99231903-99231996 94bp CCTGAAAACTCTACTCTCTTTAATCC GGAAAGACTGTATGTACTTGGCACT
Mut= 83 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTGGACAGTTAAAAGTAACCAGCAGACCCTTCTTTGGAAGTGAGGGTCTGAGGGCACAGGCCTCCCAGGATAACATCATTTCAGAGAAACTTCAGTTCTTGGGAGGGAAGGAAGGACACGTTTGGGAGCGCATCCATgcgcattcacaggcttatctgtccacgttgcttccactgtagagttgctacagcgaacggaagaaaacacggaacttggaggcgtacgtagaatggtttaatcgtctaagctacttggttgctacagaaatctgcatggtgagacaacacattgttttttcctcagatccagttttcctgctttatcttctatctgttgggcctgtgtgctgggttcctgtctgctcacgctcgtttttcttacagcctgtgaagaagaagcaccgagcgaggatgatcgagtatttcatcgatgtggctcgagagtgtttcaacattggaaacttcaattctttgatggcaatcatctgtgagtattcgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtataaacactagttttatggggcttggaaattgtatccactgaaaatctgcctttcctgaaaactctactctctttaatccagagttattatgaaacgttcagaatgctttatGGACCTAAACTAGTGCCAAGTACATACAGTCTTTCCTTAAAGGACATTCATTGAATGTCTTTAGTTCATCTATGCTCAGTTAAATATCCATCTGTAAAAGCTGAGAACTGAAACACTTGTCTAAATACAAGGTTTCTAGAGGTAT
This mutation is a 501 bp deletion beginning at Chromosome 5 position 99,231,939 bp and ending after 99,232,439 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42929 | CCT GAA AAC TCT ACT CTC TTT AAT CC | Wild type Forward | A | |||
42930 | GGA AAG ACT GTA TGT ACT TGG CAC T | Common | A | |||
42931 | TTC AGT TCT TGG GAG GGA AG | Mutant Forward | A | |||
42932 | Fluorophore-1 | AGA GTT ATT ATG AAA CGT TCA GAA TGC | Quencher-1 | WT Probe | ||
42933 | Fluorophore-2 | AGC GCA TCC ATG GAC CTA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42929 | 0.40 uM |
42930 | 0.40 uM |
42931 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |