Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:111841642-111841778 137bp GCTTAGAGTTTGGTGGGATAAAC TCCCTTAGTGAAGACATCTAGTTTG
Mutant= 147 bp
Wild Type = 137 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
GAACACAGACATACACATAAATAAATGAATAAATATCTCTAAAAAAATTAAATCTCAGTTTACCGTCTCCATCTCTCTTACTGTGTTATTAGTAATCCCAGATGTAAACTTTGAGGTGTGTAAGGTGatccatggattggggagaggcagtgagtgacttgcattctgtcttgtcttccttttcctgggtggctgttggtgagtaggaaaaggaaacaaagtggcctcttgttagaagaagaggaagatggcaaggaagactctggaggtagtagcagtgaagaagatgaagaggagcaagaaggaggccttgggtcagagaatgcaaggaagaagaaggaagatgagttgtgggccagcttccttaatgatgtaggaccaaagtcaaaagcagccccaggttcacaaactaaggtaagcttagagtttggtgggataaacacctttccagaaacgccctcacttccctgggagcTTCCTATTGTTTTAATTCGCAAACTGACTTGAAATTTGAATCTCAAATCAAAAAGCAAACTAGATGTCTTCACTAAGGGAAAAGCAAAGCATTGCTTTCTTTCTTTC
This is a 348 bp deletion beginning at Chromosome 8 position 111,841,722 bp and ending after 111,842,069 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52434 | GCT TAG AGT TTG GTG GGA TAA AC | Wild type Forward | A | |||
52435 | CCG TCT CCA TCT CTC TTA CTG T | Mutant Forward | A | |||
52437 | Fluorophore-1 | CTT TGA GGT GTG TAA GGT GGA TTC C | Quencher-1 | MUT Probe | ||
52674 | TCC CTT AGT GAA GAC ATC TAG TTT G | Common | A | |||
52677 | Fluorophore-2 | TTT CCA GAA ACG CCC TCA CT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52434 | 0.40 uM |
52435 | 0.40 uM |
52674 | 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 |