Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:119234662+119234812 151bp CAGCGCATCCCTATCAAAGA GCAGTGGCATTGTTACCTGA
Mut = 151 bp
Wt = 151 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGAGAAGGTGTTTAGATGGGAGAAGAGAAAGTGGTCCCCCACAAGGCCCTTCCTGGAAGCGCAGAAAGTGAAGTCTAGCCCCTGTGAGGAGTGTGTTCAGGCTACTCTTTCCAGCCCCTGGCATTTGGGCAGCAGAGCGGAGGGTCTAACAGACCCATTCACAACCTTGATCAggaggggacacagttaaactcacatacaaactatagccgtgttcactcatttgttcctgacattctttacgtaggtgagagccccagcgcatccctatcaaagagacttcccaatgggggcacgacaccgattcagactgagcatcatgggaagtctgactccctcggacacggacgatcttcccctggaaacggacgagccgcctcagcaagagtcaggtaacaatgccactgcctgcgcccgcatgttatttacaaataacagggggggggggtgctggggagatggctcgggggttaagagcaccgactgctcttccaaaggtcctgagttcaattcccagcaaccacatagtggctcacaaccatctgttgtgggatctggcgccctcttctggtgcgtctgaagaaagcaagagtgtacttacataaaacaaatctgagaggggggggggggaagaaaactaggaggatccacctagcctcgtaAGAAATGGGACAGTGGTTCTGAGTGCAGCTCACTGGTGGGACTTGCTTGGCACACACAAGGCCCAGGCTCTGGTACACAGAAATGGATGCAACACTTCACACCAGAGTCCCAGCAGTGTGAAACCTGAAGCAGGAAGACCCTTGAG
This mutation is a 489 bp deletion beginning at Chromosome 11 position 119,234,578 bp and ending after 119,235,066 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51138 | CAG CGC ATC CCT ATC AAA GA | Wild type Forward | A | |||
51139 | GCA GTG GCA TTG TTA CCT GA | Wild type Reverse | A | |||
51140 | CCC TGT GAG GAG TGT GTT CA | Mutant Forward | A | |||
51141 | GTG TGT GCC AAG CAA GTC C | Mutant Reverse | A | |||
51142 | Fluorophore-1 | CTG AGC ATC ATG GGA AGT CTG ACT | Quencher-1 | WT Probe | ||
51143 | Fluorophore-2 | ATC AAG AAA TGG GAC AGT GGT TCT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51138 | 0.40 uM |
51139 | 0.40 uM |
51140 | 0.40 uM |
51141 | 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 |