Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 123 bp
Wild Type = 122 bp
chr4:11303593+11303714 122bp ACAGAGACCCTCGATTCTGC TTATGGTTGGGATCAGCACA
Wt Sequence (deletions in lower case and insertions with carrots ^g^ (g insertion):
CCTCCTGCCTCAGCCTCCTCTGTGCTAGGGTTGTGGGCCTATACCACCATGCCTGGCTTTTACTTTAGAGTTTGACATTCAGTTTATTGCCAGTCCCTTCTTACTGTTCctgct^aa^GTTTGTAGGGTATTCTTTGGGTTTTCCTTCTAGAATTTCCTTGTATGTAAAACATATAATGttatatcagttgtaaaccatttgaatgttttaacaaaaatgattgctaggctattgagaaatttaatcttattaatacagtagtttaggaaattttaaatatgtccttgaatacaactgtaaatgtcagtttaaaatgtgaaggaaacaaacgaaaccaaacacaagaggctggaggagaagaaagagtgcagcctgacctttactgcttgtgtcactgcaggagctcgagcgagagatcaccttccagggagacagcgctatttactactcctactataaagacatgctgaaggcgccgtcattcgagagaggtatggccagccacagcggtaactgcagcaatgatgttgttctaaaggaaagcactcttctcaaaggttatgagctttaggatgcgggatatgatagtaagttgggaagatgcagcatctactgtaacatctgagtgcctttttttccatattatttgtgattactaatgaatgacatgttgaatcactgtcggggcagaacagtaatctggagaaaaatcatgtatttaggtgtttatgagctgacgcacaataacaaaaccatctccctgaagactatcaacgccatgcaacagatgtctttgtacccagaacttattgccagtgttctctatcaagcaactggcagcaatgtatgtatttattattattattttttttattttagatttacttcatgtgtatgagtgttttgcttgcatatctatatgtgactgcatgtgtgcccagtgcccttagaggacagagagcatcagatcctctaggacactagttctcactctgtgatattctgcatatcaggtgtttacattatgattcaaaacagtagcaaaatgacagttatgaagtagcaatgaagtaagtttatggttgggatcagcacagcatgagggactgtcacagcactaggaaggctggaagccactgctccagaagttgtagacagttctgagccactgtgggggagcagaatcgagggtctctgtaagaagaaaagtgctcttagccacggagccctccctccagccctgtgtcttcactgtgacgtctgttttcctacgtgggtttgtatctttgggttgtttaaccatcctctgtcactccctttctttttactttaggaagtgatcgaaccagtgtatttctacattggcattgtgtttggattacaaggaatgtatgtcacggctttgtttgttacaagttggcttatgagtggcacgtggctagcagggatgcttactgttgcgtggttcctaattaacaggtatgcaaggtttttacattgatatttacagattttgttttggttatagagttccaaaaatatccccattcttacagtgctcagctgttatctgcaaacctattgattgttatgtttgatggtgacgattaggtaggactccacacagggactaacttagatacaggatcacttacagagccggtctaagcgcttgCTGTACAAGTATGTGAGCTGGAGTTCAGACTCCAAGAATCCACCCTTCTCAATAGTCACAAATGATATAAAATACTTTGGCGTGACTCTAACTAAGGAAGTGAAAGATCTGTATGATAAGAACTTCAAGTCTCTGAAAAAAGA
This mutation is a 1497 bp deletion beginning at Chromosome 4 position 11,303,116 bp and ending after 11,304,612 bp (GRCm38/mm10). 61 bp before the deletion site there is an indel with 2 bp insertion (AA) and 5 bp deletion (CTGCT).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66475 | GGG TGG ATT CTT GGA GTC TG | Mutant Forward | A | |||
66476 | Fluorophore-1 | TGC TGT ACA AGT ATG TGA GCT GGA GT | Quencher-1 | MUT Probe | ||
67927 | CCC TTC TTA CTG TTC AAG TTT GTA G | Mutant Reverse | A | |||
67928 | TTA TGG TTG GGA TCA GCA CA | Wild type Reverse | A | |||
67929 | ACA GAG ACC CTC GAT TCT GC | Wild type Forward | A | |||
67930 | Fluorophore-2 | CTG TCA CAG CAC TAG GAA GGC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66475 | 0.40 uM |
67927 | 0.40 uM |
67928 | 0.40 uM |
67929 | 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 |