Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:5213099-5213198 100bp AGTGAACTGCAGCCAGGAG TCCCTTCCACGAGCACTTAC
Mut= 121 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGTGCAGGGAAATGAACCCAGGTCCTATGCAATGCAGTGTGTCCTCTTAAGTGTTGGGCCAAGTCTCTAGCCCAGGTCAGATGTGACTTGAAGTTTCAGTCCAGAACAGATACATTGTAGTAATCTATGTATTGTCATTGCAGGTGAATCAAACATCCGttctggaacaacacagagtgggaaaacattacagagtaagagtcagtttaggaccattgcaccaaaagttgtacctaaggtcctaacatccagagtgcttccctgcccttcctcaatttctgaccaagggagtctgactctaatgcccaagcctctgggcatgcccacccagaactatgctctgatgcaggtggctggccaagaggggacgttttctcttgttgctttcccaaatgttgcctcagcccagccagttcagaagcccagaatgtccacacatgaaaaccttaaactgcctattcctcgataccaaccactaagtagccaaggattaagaaagaaacaggacctgagctctcccaagagtggctccagtgaccctcctggcccatcccagctatgtcatttgacctcttccccacaagcctatcctgatcctcatcacaaacctagcctgtggaagcaaatgccaactttaaacccttctcccaccaacatcaacacagctacactgaccagtggtgttggccaaggagatttgagtcctctagtgaccaacagctgtggcgatcttgagctttctgccatgccagtgtacactacagaggactcttcttcccaacagagcctcccagcaagtatgcagaaggcaggttgtgccaggaaggaggcacccactacacctgctattgctagtgaagaactcaaagaacagttagcttctgcacagactgtcttgagcagtgcagttcagttgctctctgtagtacccaagggtaaactgcccatcctgcccttaccaagagtgaaaactacagaggtgggcaaagtggaatcagatgctgataatgcagaattgtctttatctggatgcagagcaaactgtgatgagagactgtccatcacagaagggtttgatgcagccacagaaatagccagcaagatagctgctttgcatgggtctaagcagagtgcatgtgaaagtgccttctgtcctgtcaccaaattggatctcagccacaaggcaaaacccagcagtggaataaagagaagaggaagaaaatggaaagtgccagatgagattttagcattgcagggcaaaaggaggaaatgcattattggtatgtgtggagatggcatagaaagagcaagaaacagtccccaagaagccagagaccagaagcccagggttgcttcaaggaagtatcgtaacatcatgcccaagcctgtccttgtcctgtctgctttggcacccctggcgtctcatacggctgtgctgtctcaggctcccagcagcctaggtcaagatgttctgaataatgccctgccttcaaaatgtcttggctccaagcaaagtgacagctctacccctaagcccagctctgtactccgaaatggattctctggcattaaaaagccctggcacatgtgtccagtctgcaactaccactttcaattcaaacaccaccttctagaccacatgaatacacacaccaacagacggccttatagttgcgggatctgtcgcaagacctatgtgcgccccggcagcctgagtgcacatatgaaacttcaccatggtgacaaccaccccaagaaactagtgtgctgtgaattttgtgcaaaagtatttggtcatgtccgagtctatttcggccatctgaaggaggtgcacagggttgtgatcagcacagagccctcccccagtgaactgcagccaggagacacaccaaaaaacaagaacagagatcctggtATGCAAGGACCTGACAACTCACTGGAGAGGTAAGTGCTCGTGGAAGGGAAGCTGACAGGAAAGTCTGCTGTAATATGAGAAGGAAGACCAACTCGTGGATATTTCATTCCTCCCTAGCATAGGGAATAAAATATCCATGGAAGGAGTT
This mutation is a 1756 bp deletion beginning at Chromosome 18 position 5,213,148 bp and ending after 5,214,903 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42679 | AGT GAA CTG CAG CCA GGA G | Wild type Forward | A | |||
42680 | TCC CTT CCA CGA GCA CTT AC | Common | A | |||
42681 | TTG AAG TTT CAG TCC AGA ACA G | Mutant Forward | A | |||
42682 | Fluorophore-1 | ACC AAA AAA CAA GAA CAG AGA TCC T | Quencher-1 | WT Probe | ||
42683 | Fluorophore-2 | CAA ACA TCC GAT GCA AGG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42679 | 0.40 uM |
42680 | 0.40 uM |
42681 | 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 |