Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:33410973-33411090 118bp GAGTCCCCCGAAAGAACAG AGTCAGGCGCATCAGACACT
Mut = 119 bp
Wt = 118 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GCAGACCGTAGTGCTAGCTGCAGAGTTGCAGTGAGCTATTTATGCTGCCTGCTCTTACTGAGGAGCCCTGGCGAATTACGGGTGCTCTTTGTCTCTGCAGATTACGTTTCGTGTCGGGagaaatggacaacagcagtttcattcagtttgatgtgcctgagcacagcagcacggttctgagccaactaaatgagctccgcctgcaagggaagctatgtgacatcatcgtccacattcagggtcagcccttccgagcccacaaagccgtcctcgcagccagctccccgtatttccgggaccattcagcactgagcactatgagtggcttgtcgatctctgtgattaagaaccccagcgtgtttgagcagttgctctccttctgttacactggaaggatgtccttgcagctgaaggatgttgtcagttttctgaccgcagccagctttcttcagatgcagtgtgtcattgacaagtgcacacagatcctagagagcatccactcgaagatcagcgtgggagacgttgactctgttaccattggtgctgaagagaatcctgagagccgaaatggggtgaaagatggcagcttcttcaccaatcctgttgaaatctcccctccgtactgccctcaggtacggcagcctccagtcagcagtgatcttcggatggagacaacccccaacaaagcccttcggagccgcttgcaggaagaggggcactcagatcgggggagcagtgggagcgtgtctgagtatgagattcagatagaaggggaccacgagcaaggggacttgttggtgagagagagccagatcactgaggtgaaagtgaagatggagaagtctgaccggcccagctgctccgacagctcttccctgggtgatgacggctaccacacggagatggttgatggggaacaagttgtggccgtgaatgtgggcgcctatggctctgtgctccagcatgcatacccctactcccagacggcttcacagccttccagtgtgccagaagcttttggaggtcagaccaattccagtccatccaggtccatgctgagctgcttccgaggccgtggggcccgccagaagcgagctctgtctgttcacctgcacagtgacctgcagggtgtggtgcaggggtctgacagtgaagccatgatgaacaatcccggttatgagagcagtccccgggagaggagcgccagggggtattggtacccatacaacgagaggttgatctgtatttactgtgggaagtcctttaaccagaagggaagcctcgacaggcacatgcggctgcatatggggatcaccccctttgtgtgcaagttctgtgggaagaagtacacgcgcaaggaccagctggagtatcacatccggggccacactgatgacaaaccattccgatgcgaggtctgtgggaagtgctttccattccagggtaccctcaaccagcacctgcggaaaaaccacccgggggtcaccgaaggcaggggtcgcatggagtcccccgaaagaacagacatgtatgtggaacagaaactcgaaagtgatgcatcagcctcagagatggcactagattcccGCCTGGAAATGCACACAGTGTCTGATGCGCCTGACTAAGGAGCACCTGCACAGAGCACAGCAATCAGCGCGTCCTTGTGATCTGCTTGGTTGTAATCTTTGCTTTTCCCCACCACCTGGACATCTCTCGGTTTTTTTGCTAGTTTCTCTGAAGGTTCTGGGTGGGGACTTCTTGTGTTTACCCTCCC
This mutation is a 1469 bp deletion beginning at Chromosome 2 position 33,411,009 bp and ending after 33,412,477 bp (GRCm38/mm10). There is a 61 bp inverted insertion (GGGAATCTAGTGCCATCTCTGAGGCTGATGCATCACTTTCGAGTTTCTGTTCCACATACAT) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51214 | AGT CAG GCG CAT CAG ACA CT | Common | A | |||
51215 | GAG TCC CCC GAA AGA ACA G | Wild type Forward | A | |||
51216 | GCA GAT TAC GTT TCG TGT CG | Mutant Forward | A | |||
51217 | Fluorophore-1 | AAC AGA AAC TCG AAA GTG ATG CA | Quencher-1 | WT Probe | ||
51218 | Fluorophore-2 | CTG TTC CAC ATA CAT GCC TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51214 | 0.40 uM |
51215 | 0.40 uM |
51216 | 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 |