Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:100702981-100703100 120bp GGCCATTGATCACATCCTACA AGCGGAAGAGACGAGCATAG
Mut= 121 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGATGGCTCGGAGCCACGACCTGCTCATCTGTACGGCAGAGTTGTTACAGTTGGCACTCAACAGCTCTGAGGAGGATGAACACGTAGAGCTCAGAGGTGAGGGGGAAAGCCGTGACCTGctcacaggacgtgctcctgggcaagcaagcctccttcctctagcccagcctcgctttcattcctacctgctccctgtctgggacccagcccaaagtagggaagggaagaggaaaacttcagagaaaaatgagggcttcacaaaagtgctatgagaaagaagcagcagcagcagccgtccactgtctctcatgtgcccccagaattctcgctgattgtggtggacgagtgtcaccacacccacaaggacaccgtctacaacaccatcttgagccggtacctagaacagaagctgaagaaggcagagcccctcccccaggtcctgggtctcacagcctccccaggcactggaggggccaccaagctccaaggggccattgatcacatcctacaggtcagctgagcccctcccaccatagatctttgcttgtcttgcctcccagtCCTACCAAATATACTCTTCATACTATGGCTATGCTCGTCTCTTCCGCTACCTTGAGCATATGAGGCTTGTGGGACCCTTTGAACTTACTATTCCTTTTGCCTGGAAATGCC
This mutation is a 442 bp deletion beginning at Chromosome 11 position 100,703,029 bp and ending after 100,703,470 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42134 | GGC CAT TGA TCA CAT CCT ACA | Wild type Forward | A | |||
42135 | AGC GGA AGA GAC GAG CAT AG | Common | A | |||
42136 | ACA GTT GGC ACT CAA CAG CTC | Mutant Forward | A | |||
42137 | Fluorophore-1 | AGC CCC TCC CAC CAT AGA | Quencher-1 | WT Probe | ||
42138 | Fluorophore-2 | CGT GAC CTG CCT ACC AAA TAT AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42134 | 0.40 uM |
42135 | 0.40 uM |
42136 | 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 |