Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:152186249-152186338 90bp AGCGCTGGCTTTTATCCAGT TGTCATCCAGAAGAGAGCTATAA
Mut= 92 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TTAAAACTATTAAATTTGTAATACTTAAGTATGTTAGAACAGGAGTTTTTGAGGTAAGGGAGTCTCAATTTCCTTTTATTTTTCATATTTATTGTGAAACAAAAGATTCAAGAAGAAATTATTAAAGCAAAGAATTTGGAGGAATGATTTAAAAGCTAAAACGTTAACataggatggatcttcagaaactattctgtttaattctaatgatgtttcaggccgtaagtgataagtcagagtttattagcagggcaatctgtaagtggtcagctgtctagactgccggcccttttcctaatcttgcttatgtcttcaccaggcttgaagggaggcgctggaggtactgatggacaaggaggtaccttcaggtacactttccacggagatcctcatgccacatttgcagcctttttcggaggttccaacccttttgaaatcttctttgggagacggatgggtggaggtcgagattctgaagaaatggaaatagatggagatccctttagtgcctttggattcagcatgaatggatatccaagagacaggaattctgtgggaccctcccgtctcaaacAAGACCctcccattattcatgaacttaaagtatcgcttgaagaaatatatagtggctgcaccaaacggatgaagatttctcgaaaaagattaaatcctgacggaaggagttacagatctgaggacaaaattctcaccattgagatcaaaaaagggtggaaagaaggcaccaaaatcacttttccgagggaaggcgatgaaacaccaaatagtatcccagcagacattgtttttgttattaaagacaaagaacatcccaagtttaaaagagacggatcaaatatagtttatactgctaaaattagtttacgggaggtaagtcagtaagtcagccctgagactctaagacggagagatgactgctgctgattgactagggggctgtctgggcagcgctggcttttatccagtgctctgcagacttattcctcatggtaaCTCTCTAGGAAGTGGTGCTATTATAGCTCTCTTCTGGATGACATCATAGTTTCTAAATTTATTTACTGATGTTAACTATTTAAATGTCGGTTTTCAAGTACAAGTTTGGTTAGCTACTTTATGGCCAGTGTTAGGTTAACTCTAGTTTTAGGTTGTGGTCTGTGAGATCCGCA
This mutation is n 837 bp deletion beginning at Chromosome 3 position 152,186,292 bp and ending after 152,187,134 bp (GRCm38/mm10). Also, after the deletion of 381 bp of sequence there is a 6 bp [GGTCTT] endogenous retention followed by an additional 405 deleted bp.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43718 | AGC GCT GGC TTT TAT CCA GT | Wild type Forward | A | |||
43719 | TGT CAT CCA GAA GAG AGC TAT AA | Common | A | |||
43720 | AGC AAA GAA TTT GGA GGA ATG A | Mutant Forward | A | |||
43721 | Fluorophore-1 | CTG CAG ACT TAT TCC TCA TGG TAA | Quencher-1 | WT Probe | ||
43722 | Fluorophore-2 | AGC TAA AAC GTT AAC AAG ACC CTC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43718 | 0.40 uM |
43719 | 0.40 uM |
43720 | 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 |