Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:181575225-181575324 100bp TGAATAATGAGGCCCTACCC AATTAATGGCAAGATGGCAGGA
Mut= 116 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CATACATCTTTCTACTGGCATGTGGCCAGTATACTGCACTTTCACCTCCTGGGGCAACCAGGGGTAGGGCAGCGGTGGCTGGACTACCTAAGGAGTGTGCTGTGCTGGGGAGAGAGGCACAGTCCTTAGATCCTCCTTTTTCTTGAATAATGAGGCCCTACCCACCACAGTCcacatgggtaggattgtggctctgagatcatgcatatcctcctgtgtcctcctgccatcttgccattaattgctgtcccttgttcttgctgtgtgaacagcctgagatcttgtgccccagttaaccctatggaacaggtaaaggcaggtttcttggacacactctccccagttgagcctatattgtgcctgtcccactttggtctgaccagagaggttgaaaactgttgactgaggctaagcacattcctctttacagcagcaatgcagggaccttggacagacttctcccaccagtgggtactgggcgctcaccccggaagcgcaccaccagccagtgcaagtcagaaccacccctgcttcgcacaagcaaacgtaccatctacacagctgggcggccaccatggtacaatgaacatggtacacagtccaaggaggcctttgctattggtgagagtggtatgggaaggcaccttggggatctgtctgcatggccagcccagctgcaacagtttgcaaggtaccctttcactccacaggcttaggaggtggcagtgcatctgggaagaccactgtggccagaatgatcattgaggcactagatgtgccctgggtggtcctgttgtccatggactccttctataaggtgaggccctgctcatctacccccagagcatcttctctctgtgactgctttgcctctacaaatgtaggcctgtggtcagcagcaggcttcatcatgcccctccacccccaggttctgacgcagcagcagcaggaacaggctgcctgtaacaatttcaactttgaccaccctgatgcctttgacttcgatctcatcatttccacccttaagaagctaaaacagggcaggagtgtccaagtgcccatctatgatttcactacccatagccggaaaaaggactgggtaggcccatggtgtgggggcctacctatgggagaggttgcaggggtctgagctggacattaaaactctctgggttccttttcagaaaaccctgtatggtgcgaatgtcatcatctttgagggtatcatggcctttgctgataagacactgctggaggtgagagcttgccgggagcacccactccacaggcatccccataaatactctcttagtaatacgccgccccatcctcacagcctctatgatttgagatttctccctattgggcagttctggatgcgccccacctggtgtatcCTAGGCATTTACAGCCGTTCTTTGCCATTAGCTGACTACTACAGTGGGTGTGGCGTCTGCGGACAGTGGTTTCCTCTCCTACCTTCGGGGAGGGAGCTGTACTTCTGTCTCCCTTGCTCTTGATATTTTTCCTTGCTAAAGAACAAACAAGGACTTAAAGCCAGTGAGTTTAAGGGACTTTGC
This mutation is a 1225 bp deletion beginning at Chromosome 2 position 181,574,071 bp and ending after 181,575,295 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46202 | TGA ATA ATG AGG CCC TAC CC | Common | A | |||
46203 | AAT TAA TGG CAA GAT GGC AGG A | Wild type Reverse | A | |||
46204 | CGA AGG TAG GAG AGG AAA CCA | Mutant Reverse | A | |||
46205 | Fluorophore-1 | AGG ATT GTG GCT CTG AGA TCA T | Quencher-1 | WT Probe | ||
46206 | Fluorophore-2 | GTC CTA GGC ATT TAC AGC CGT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46202 | 0.40 uM |
46203 | 0.40 uM |
46204 | 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 |