Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:66672417+66672508 92bp CAGAGAAGTACCTGGCAGCA CCCAGTATAATGAGCCCTTTTC
Mut= 83 bp
Wt= 92 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCTTATTCCCCTCTGCTGTGGAGTTACATGCAGAGTCCTCACCTCTAGCTATGGAATACTCTTCCTGCTTCTGTATCTTACTCAAGCTTCCTATAGGCCCAATCTGCTGGCTAATGCTAGTCAGAACCACTGAGGATTCAAGATTGTGAGGGAGGAGTTAAGAAGTATGTACGCCTCCCttttttgccagcgttgtgtatgtttctctctgaagtttcaggcctgccttgtccccaggaggatcttttgtctccctaaccaccttctaccccaagccaccctgcaactcgcattgctgcctctcctgccttgtgtgatcacaactctgactttcctgtatgacttgctagactttggcctcactgagctggagccaaaatgtggcttcagaaaggctatatggtatcacatgatatttctctcttcaggacagtccaatgccaaaatgatggtcaatcttgctggtgtgtggattctgatggcagagaagtacctggcagcaggcagctggggaggccaacagtttgtacgtaacaaaggaggcacctaatGGAAAAGGGCTCATTATACTGGGGCACCTCACATCTAGGGACAAAAACTAGACTTAAGTAAGTGAGTTGGGCAGTCGCCCTAAGGGCTGCTAGCTTCTCATCATTGAATATGTAGAAGGTCCAGTTTCCTAAAGACCACTATAAAAACCATATGAGACACTTGAGCCTAGCCAGTAGCTAGCAAGTACTTCCCA
This mutation is a 372 bp deletion beginning at Chromosome 15 position 66,672,114 bp and ending after 66,672,485 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39602 | CAG AGA AGT ACC TGG CAG CA | Wild type Forward | A | |||
39604 | CCC AGT ATA ATG AGC CCT TTT C | Common | A | |||
39605 | GTC AGA ACC ACT GAG GAT TCA AG | Mutant Forward | A | |||
39606 | Fluorophore-1 | AGG CCA ACA GTT TGT ACG TAA CA | Quencher-1 | WT Probe | ||
39607 | Fluorophore-2 | TAA GAA GTA TGT ACG CCT CCC G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39602 | 0.40 uM |
39604 | 0.40 uM |
39605 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |