Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:74119706+74119805 100bp TGGTTGGTTCTTTCATGTGC GGATGAGCTGAAACGTCATAAAG
Mut= 91 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAATTCTTTCATGTATCTCAAACCTAAACACAGCTTTGGGCAGATTGGCTTTTACGTGAGAGTGGAAAGTGTCAGAGGAGAAAATCTTCCTTGATCTGTGATCTGGAGAGTTGGAAGGGAAGGTGAGTTGTGATGTAGAGTGATGAGCTTCTTCTTGCTTTGGGTTGTATTGTACATTTGCTCTGGATATCCTGTTAAACATGTTTTTTGTTTTTGTCACCATCTTGAGCATTCTATTGGCTTCTTTCCTACAACCTCCTTCCCTTCCCTTCTGGTTGGTTCTTTCATGTGCTGAGGTTCCTATGTGTGTTTGCCTCTGTGatcttttgccgcttatctcatgccttttctttatgacgtttcagctcatccaccatacaccatgtgctttagagtgaaattctacccacatgaacccttgaagattaaagaagagctcacaaggtatttttcctttttgcatatttactccaccgcttgcattcaggtagagcctttgctatcttataggctagacattcctgttatcgatttgagaacacctccagGAAGTAGGCTGTGGAGTCAGCATTGTGTTCCTCATATTCTTGTTGAATTTGGGATACTTTATAGAGAACCTTGTTGAGAAAGTATTATCTGTACAAGAAAGCAATGGTTATAAAAGGGGAGCTCTCTAAAACATTTGACAATGGAATGCTAACAAAAGCAGAAGATGACTGTCTTTTGGGTATGGGCAAATGCAGAGAGCAGTCCTAGTCTGTCCTTTTGGGTCTAAGACTTTCTGGAAGAACAAGATTACTGGACAGAGAGGACAATTAGCTATTTTGCTTTCCACTTGCTACATGACAACTACCAAGCAGATGAACTCTCCAACTTTCTTCTCTTTCTCTCTCTCTC
This mutation is a 227 bp deletion beginning at Chromosome 4 position 74,119,755 bp and ending after 74,119,981 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48267 | TGG TTG GTT CTT TCA TGT GC | Common | A | |||
48268 | GGA TGA GCT GAA ACG TCA TAA AG | Wild type Reverse | A | |||
48269 | CAA GAA TAT GAG GAA CAC AAT GCT G | Mutant Reverse | A | |||
48270 | Fluorophore-1 | ATC TTT TGC CGC TTA TCT CAT G | Quencher-1 | WT Probe | ||
48271 | Fluorophore-2 | TCT GTG GAA GTA GGC TGT GGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48267 | 0.40 uM |
48268 | 0.40 uM |
48269 | 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 |