Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:4904759+4904890 132bp AGTATTCAGCAGGCATAGATGG AGGTGAAGAGCAGTAGGGAGA
Mut= 133 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TAAAAAACGATGTCCACCATTTGTTCCTATAGTATTTTATTGAACAGATGTCATTGAGTACCTGTTTATTTGCTATACCTCTGGGACTTGGGAAAAGCTAGATGGAATATATGGATATTAGTGAAGTAACAATCCAAAATTGCTTATTAGTATTCAGCAGGCATAGATGGCTACCTAGCTAGTGGTTTCCTTCAgacccacggcaccttgattaatgatatttaggctttcagctgaaggaagaagagcaagaacataatctccctactgctcttcacctatctaagccagtaagtcctggcatgctttttatggatgacggaaagagtcaggaccacccacatgaaaagtgaatggtgccttttcctcttgtgcccttcactgcagtgtctcagagcatacccttcatgatgatgaccctgctcaccatgcagaccatgctcaggctggtagaggatgaaaatatgaggcaaaccttctgtataggtgggtattcctatgatttgctttctgctgaaaaataaacatgcttccaggacatttctcacatggtgcagatagatcacacagagtttccaaaagagtttatatcaaatcttcgctgccatggatgtgtatgagtaccacctataacttagtctagttgacatgtgtgtggcattggccgtgttaaatatgttccattctagacagactacctcctagtaagagtatataataacaatcttaatttatactctattcactactaattgaaattggctaaataattttcatgttttcatggttatttgatgaagttttaagatgatttagaaacttttgctaattgtctgtgctttcttttagaaaaatttgatgtttctttccatcttcacctttgtttcagtctgtggttgtattttttttatttttaatgcagcccttgaaaatttcagcaagtccatttacaagtatgtcaaccactggaaggattttccatatcccaaattagatgctaaccggctctctgacaagatcttcatgctgttccggtatataatggagaaatgggccccgcaggcctcacccatgcatgtgagtgaggtttcagataggctcaagttgtaaatccccatactggtcaaaccagttttagagtttggttctatcttatatatgtgcatcttacacgcatgcgcgcgcacacacacacatatgttctaaacatatatatatatacacacacacacacacacactcatatgtgtcctaaaatttagtgaacttctttaggttcttgccaatactttaaGCTATTAAAGCAGTTTTTAAAAGAATATTTCCCAGTTAAATCATCAAATATGGAACTTCTTATGGGTATCATTAGTTATGTTTTGGGAGGTATATTAAGTCTACCAATTTTTCTATCTATTTCAAGTCACTGTGTATCCATTGTCCCACTTGAAGTTTTTGAAGAACAACAATATA
This mutation is a 1100 bp deletion beginning at Chromosome 15 position 4,904,805 bp and ending after 4,905,904 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44820 | AGT ATT CAG CAG GCA TAG ATG G | Common | A | |||
44821 | AGG TGA AGA GCA GTA GGG AGA | Wild type Reverse | A | |||
44822 | CCC AAA ACA TAA CTA ATG ATA CCC | Mutant Reverse | A | |||
44823 | Fluorophore-1 | CCC ACG GCA CCT TGA TTA | Quencher-1 | WT Probe | ||
44824 | Fluorophore-2 | TTC CTT CAG CTA TTA AAG CAG TTT TTA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44820 | 0.40 uM |
44821 | 0.40 uM |
44822 | 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 |