Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:24220325-24220425 101bp TGCTTTTAGACTCCGGTTGC CAGCCATGGTATGGTCCAA
Mut= 81 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCTGCAGGTCTGGTACGCGGACCCAGGACCTCCTGGCCAGTCCCTCCTCGGACCATCAGGATTATGCAGAGTCCCAGGGCCTAGAAGGCCAAATACAAATGCATGCTTTTAGACTCCGGTTGCCATTGCTATCCTTACCCCttatccagggtctgaggctgggaatctgcttagggatagaatgtttggaccataccatggctgccctgtctttgcagggaaccagccaggaccccatctccagttccagaaaccaaagaagaggacccaagtccaggtaaacgttagctgctttttatgggtgctgtccctggcctttctctggcttgctgtcatgggggaatccatcctgggacagtgaggaggaattcttgcatggttcttgacgttcatagcatgtgcaccgagttctcaagatctcgaggagctggaacttttgacccaggctctggagaaggctgtacgtgtgaggaaaggcgtgtccaatgctggacaaagagacagaacccccactctgacatctaaggcagccacttctggtgccgctgccgcctcccatcctcgggctcccagccggggtggcagccgtgtattgggtacaagatccaccaagggtatacagcgggccacagcccctcccaaggattaccctgagcacagactacgatcaaagggtgataagacccatgtaagaacacaagaccaaactactgggtatggaccagacctcagggaccaacaaatgaccccgtcatctgctcatcacaccacagaactcttcgcattaaaggagaaggggtaagcttcccagacgctcgtatcctgacagggcattgagctggttgaggaagcccctggtgccgacccgtaagggtcatgcactgaagcaaacagggcagaacagggttgggatggtatcaggtttgtcgttctggagcctaaccagcccatgagctctctggactgggcgagcacccaccctcctccagaagccgttttcaaaaagcttgtcagaattaagctgttaaaggagaggaggtgagaaggttcgccccaatgggTTCTGAGCCATGAGTAAGAATGACAGTCCTAATGCCGGGCTTGGTGGCCCACGCCTTTAATCCCAGCACTCGGGAGGCAGAGGCAGGCAGATTTCTGAGTTCGGGGCCAGCCTGGTCTACAGAGTGAGTTTCAGGACAGCCAGGGCTACACAGAGAAACCCTGT
This mutation is a 920 bp deletion beginning at Chromosome 17 position 24,219,468 bp and ending after 24,220,387 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41039 | TGC TTT TAG ACT CCG GTT GC | Common | A | |||
41040 | CAG CCA TGG TAT GGT CCA A | Wild type Reverse | A | |||
41041 | CAA GCC CGG CAT TAG GAC T | Mutant Reverse | A | |||
41042 | Fluorophore-1 | TGA GGC TGG GAA TCT GCT TA | Quencher-1 | WT Probe | ||
41043 | Fluorophore-2 | CCC CTT CTG AGC CAT GAG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41039 | 0.40 uM |
41040 | 0.40 uM |
41041 | 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 |