Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:155731509+155731619 111bp CACTAGGCTGGTAGACTTATGCTG GCTTGCTTCTCACAGGTTGTC
Mut= 98 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GACCATCCGTCGTCTAAGGTCTACTCTGTGCTGCTTTGGACACTTAGAAACAAGCATCTGTGTGGCCTGATCTGTCTTGAGAGGAGACAGTCCCTGCCATGCCTCTACTTTCCAGTTACAATGGCAAGGCTTTCTGGAGCCATTCTGTGGGTTTGGGTTATCGTGGGAGTTATGATCTctctacgaagtcagcaccttccctgtgcttcccatgttagggcagtggtgaccctgcttgtgttcaggcctagagctatgttgcccctgttgacaatggccaacttggaaccccctagaggcaaggggccattgacaggtctaggttagtaactatctttaatccaaagctccttgactctggtgttcagctatacacaaaatggaattttacatatgttggacagaaataaaaggatcaagccccgtccagaaaggttccagaactgcacggacctgtttgacttgatcctcacttgtgaagagagagtctatgaccaggttgtagaaggtgagtgcacagagcctgacctgctgttcaggttgtaccaccaacactaggctggtagacttatgctgccctgggcgacagtgatggagacagagggtgcccccacatcCTCTGACCTGTATGGACATCTGGAGGACAACCTGTGAGAAGCAAGCAGAAGACTGATGTCCAGCAAAACTCAAGCCTTGTGGCCTTCAATGTTTGCTTTTCCTATGTTAGGCCTGACATTTTATAGAACATGCTATTGGTGCCAATACTGACTGGCATAGGATGGCTTGAGGATTCTATCTAGGATTCTTA
This mutation is a 439 bp deletion beginning at Chromosome 4 position 155,731,135 bp and ending after 155,731,573 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44545 | CAC TAG GCT GGT AGA CTT ATG CTG | Wild type Forward | A | |||
44546 | GCT TGC TTC TCA CAG GTT GTC | Common | A | |||
44547 | AGG CTT TCT GGA GCC ATT CT | Mutant Forward | A | |||
44548 | Fluorophore-1 | CGA CAG TGA TGG AGA CAG AGG | Quencher-1 | WT Probe | ||
44549 | Fluorophore-2 | CGT GGG AGT TAT GAT CTC TCT GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44545 | 0.40 uM |
44546 | 0.40 uM |
44547 | 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 |