Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:4472959+4473068 110bp CATCCAAGCCTCTCTTTGGA TCACGTCATCATCTTGGGTCT
Mut= 96 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCCTCTATTTCTCGTGGAACCCCATGAAGTACTCATGCACCATGACAACCAGTTTTATGCTGCTTGGATGATTAAACACAGGGCTTTGTGCGTGCCAGGCAAGAACCTTCCTTCCAACTGAACCACATCCAAGCCTCTCTTTGGATTTTACAAAAGTCATTTGTGTTTCTGCAGTGTGCCTGAGTTCtcaggcatttcactgagaacatgctagagacccaagatgatgacgtgacttagcaaaagtctctcgtgagttaatcttcccttctgtgttcttcttccagcccaagcttttggccaaggagcttcttgacctcgtggcctctcatttcaacctgaaggagaaggagtactttggaatagcattcacagatgagacgtaagtgctgtccagagcccacagaccccaaacaaaagagcaggatgtcaaattaccctctctctgctgtgcacagcaaaatcaggctgcaggccacttgggtcccatgggggctacagggtcagtccaagttcaccatctgaatttcaaaagccggtgATCATGCCTGGAGTGGCCAAAGGATATAATTAGATAGATGCTAATATTATGAAATTATTAGGTAATGTTCTTTAACCAAGCAGATTTGTGTTCAGATGAATGTCTAAAATGTTTATTTTTTTTAAAGAGTAGATCTGTGTGGGTTTAGTCTTTATTAAGGTTAAATGAAAACTTCTGGAAAGT
This mutation is a 354 bp deletion beginning at Chromosome 2 position 4,473,021 bp and ending after 4,473,374 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40535 | CAT CCA AGC CTC TCT TTG GA | Common | A | |||
40536 | TCA CGT CAT CAT CTT GGG TCT | Wild type Reverse | A | |||
40537 | TCT AAT TAT ATC CTT TGG CCA CTC C | Mutant Reverse | A | |||
40538 | Fluorophore-1 | CAG GCA TTT CAC TGA GAA CAT G | Quencher-1 | WT Probe | ||
40540 | Fluorophore-2 | AGT GTG CCT GAG TTC ATC ATG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40535 | 0.40 uM |
40536 | 0.40 uM |
40537 | 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 |