Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:56676330+56676447 118bp GTATTGGGTTTTGGGTTTCA CTACACACGCCTGAGCACAC
Mut= 119 bp
Wt= 118 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAAGGGCTTTCACGGTGACATTTTCATACCTGTATATAATGAATTGCAAGGTTTTTTTTGTTGTTGTTGTTGTTTTATTTGAAATAAGGTCTCACTCCATGACTGAGCTGGCCTTGAATTTATGTAGATCTGTCCCTGCCATCCTAGTGCTAAGATTAAAGGTTTACACTATCATGTAAATGTATATTCAAACATTTTAAGTAATAGATTCTTAAAAGAAAATGGAGAAACAAAGTATTGGGTTTTGGGTTTCATTTCATATGGCTTAGCGTGTCTGCAGAATAGTCTTGGTggcgggagggagcagtgggagaaacattaaataatcagctgtgtgctcaggcgtgtgtagaatatttggtttgcaaatattacagtatcaactctttatcttttattttttgttttgcattcattattctttaggagaaagagaaaaaaaatgaacccaaaggaaaataccagaaaaggtatgatttggacaaagaagaaaaagccagaaaagagccaaaggtattaaaatgtaagtacaaaaattaaaatagttccatgttcattgccattgttaaaaaaattattaatggattttatgtgtattatgtgtacaccagGGTATATATATGTGCATATGTATATCTGATATGTTGTAAAGGTTGGATGTCTCTTTTCTGAAATATTTGGGACCAAAAATGTTTCTGATTCCTCTTCCTGGTTCAATGAAATTTATATGTACATATAATGAGATTTCTTAAGAATAGGCCCAAGTTTAAATCCAAATTTTGTTTTATATGTACATTATATGCACTCCTTTGTAATACTATGATGTTTTTAGTGTTCCAGTGTTTTTGAGTCTTGCATTAAGAGTAAAATTTTCTTCTTAATACCTTCATACTGGCTTAAGTTT
This mutation is a 319 bp deletion beginning at Chromosome 14 position 56,676,388 bp and ending after 56,676,706 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48262 | GTA TTG GGT TTT GGG TTT CA | Common | A | |||
48263 | CTA CAC ACG CCT GAG CAC AC | Wild type Reverse | A | |||
48264 | TCA GAA AAG AGA CAT CCA ACC T | Mutant Reverse | A | |||
48265 | Fluorophore-1 | AGC AGT GGG AGA AAC ATT AAA TAA TC | Quencher-1 | WT Probe | ||
48266 | Fluorophore-2 | AGT CTT GGT GGT ATA TAT ATG TGC ATA TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48262 | 0.40 uM |
48263 | 0.40 uM |
48264 | 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 |