Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:97246452-97246561 110bp AAGAGACTCAGAGGCGACCA CTGCGATCACCAACAGACAC
Mut = 110 bp
Wt = 110 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCAAGAGACTCAGAGGCGACCATAATGTCGTTAAGGTTCCACACACTGCCCAccctgcctagagctgtcaaaccgggttgcagagagctgctgtgtctgttggtgatcgcagtgatggtgagccccagcgcctcaggaatgtgccccactgcttgcatctgtgccaccgacattgtcagctgcaccaacaaaaacctatctaaggtgcccgggaaccttttcagactgattaaaagactggatctgagctataacagaatcggactgttggatgccgactggatcccggtgtcgtttgtcaagctgagcaccttaattcttcgccacaacaacatcaccagcatctccacgggcagtttctccacaaccccaaatttaaagtgtctggacttatcatccaataggctgaagtcggtaaagagtgccacattccaagagctgaaggctctggaagtactgctgctgtacaacaaccacatttcctatctggaccccgcagcgttcggggggctttcccacttgcagaaactctatctgagtgggaactttctcacacagttccctatggatttgtatactgggaggttcaagctggctgatctgacatttttagatgtttcctataatcgaatcccttccataccgatgcaccatataaacttagtgccggggagacagctgagaggcatctaccttcacgggaacccatttgtatgtgactgttctctgtactcgttgctgatcttttggtaccgtaggcactttagctccgtgatggattttaagaatgactatacctgtcgcctgtggtctgactccaggcactcccaccagctgcagctgctccaggagagctttctgaactgttcttacagcgttatcaacggctccttccacgcacttggctttatccacgaggctcaggttggggagagggcgatcgtccactgtgacagcaagactggcaatggaaatactgatttcatctgggtcggtcccgataacaggctgctggagccagataaagacatggggaactttcgtgtgttttacaacggaagtctggtcatagagaaccctggctttgaggacgccggggtatattcttgtatcgcaatgaacaggcagcggctgttaaacgagacggtggatatcatgatcaacgtgagcaatttcaccataaacagatcccacgcccacgaggcgtttaacacggcctttaccaccctggctgcctgcgtggccagtatagttctagtgctactgtatctgtacctgacgccgtgcccatgcaaatgcaaagccaagagacagaaaaacacgctgagccaaagcagtgcccactcgtccattctcagtcctggccccactggcgatgcctctgctgacgatcggaaggcaggtaaaagagtcgtgtttctggagcccctgaaggacacggcggccggacagaatggcaaagtcaagcttttccccagtgagaccgttatagccgagggcatcttaaagtccaccagggcaaagtctgactcagactcagtcaattccgtgttctcagacacaccctttgtggcatccacttaGCTGGGTGCCTGTGTTCGTCTGGTGTCGTGATTTAAAGTCTCCCTGCTTTAACTTTGGTCCGAAGAATTAATAGCAGGACGGGGTGAAAACCCTATTTTGTCTTTTGAGATGAAACCAAATGGTGTCTTTTTAAGATGGTT
This mutation is a 1531 bp deletion beginning at Chromosome 15 position 97,244,981 bp and ending after 97,246,511 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51010 | AAG AGA CTC AGA GGC GAC CA | Common | A | |||
51011 | CTG CGA TCA CCA ACA GAC AC | Wild type Reverse | A | |||
51012 | GAC CAA AGT TAA AGC AGG GAG A | Mutant Reverse | A | |||
51013 | Fluorophore-1 | AGA GCT GTC AAA CCG GGT TG | Quencher-1 | WT Probe | ||
51014 | Fluorophore-2 | CCA GCT GGG TGC CTG TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51010 | 0.40 uM |
51011 | 0.40 uM |
51012 | 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 |