Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:38018742+38018883 142bp GACAGTCATGCTTCCATAAAAGG CTTCATTGTTTTCTGTAGGCTAAAAAG
Mut = 136 bp
Wt = 142 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GTGTGGTTGGATTTGTTCCTATTTTTTTTCCTCTTTTGTTTTCTTTTAGATTTTTGCTTTGTGTATGAGTGTTTTGCCTGCATATAGCTACATGTACCATGTGTGTGCCTGGTGCTCACAGAGGTCAGACGAGAGGGCATTAGATCCTCTACAGCTGGAGTTACAGATGGTTGTGAGCTGCATGTGAGTGCAGGAGCTAAACCTGAGTTCTTTGCAAGAGCAACAGTTGCTCCTCACTGCTAAGCTACCTCCAGACTCATACTTTAATATTCTTGTGAAACAAGACAGTCATGCTTCCATAAAAGGTATTGAGgtgagcactgagtgaagtgctccacactgaggagccccatgacctttaagggatgacagtctgttgatgtttctaataagggatactttttagcctacagaaaacaatgaagaagaatctgcctcaaaacaagataaaaagaagaagggacaaaaaggcaagaaaacaagttttgatgagaatgatagtgaagaattggaagataaagattcaaagtcaaaaaagactgccagacctaactcagaggcacccctttctgggagtgaggatgctgatgattctaataaactttctaaaaaaggtaaaaaagcccagaaatccacgaaaaaacgggacgggtcagaagaggatgaagacaatagcaaaagaagtaaagaacgttcgagagtaaactcctctggtgaaagtggtggtgagtccgatgaatttttgcagtccagaaaaggacagaaaaaaaatcagaagaacaagtccgttcctaccgtagacagtgggaatgaggacgatgactcttccttcaaaattaagacggtggcccagaagaaggcagaaaagaaagagcgcgagaagaaaaagagagatgaagagaaagcgaagttgcggaagatgaaggagaaagaagagctagagaagggcaagaaggagcagagtaaacagagggagcctcagaagagacctgaggaggaggtgttgacactcagagggactcctgacacaggagctgcctcggaagagaaaggagacactgctgcagctctggaaggttggtcatggccacagaaggctctggttcatgtttgGTCTAAGTTGTGGTCATACTATCTTGTGACTAGTCCTCATACACATGTGTGTGTGTGTGGGGGGGGGGTCCATCTCTTTGTGTGCAGAGGAGGCTGGAGGTCGATTCCGCTGTCACTTTCCATGATCTGTTTTTAATATTACACCATTTCTCTACGGTCTTTCTGTATACCTCTCTTTTAAATTTATGGCCTTGTTTTTTTCCCATGAACTGCTGTGAGGTTGAGTCCCCCGGAGTGTCAGAAACTGTATGTATAAAGCCTCACCCAAATGGCTTTCTGAACAGCAGTTGAATAGAGACACCAACAGATAGATACAGTAAAGCCAGTGCCTCAGCTCTCTGTAGAGAACCACAGGCAACCAAGGAATGCTGAGATAGAGCATAAGAAATCGTCTTCCATCTGATTTTTTTGAGACAGGGTTTCACTGAACATAGAGTTAGTATACTGGCTGGTCAGGAAACAGTGCTGTCACGTCTGGCTTTTCTTTTTATTTTTTAAGATTTATTTATTTTATGTA
This mutation is a 800 bp deletion beginning at Chromosome 1 position 38,018,772 bp and ending after 38,019,571 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48532 | GAC AGT CAT GCT TCC ATA AAA GG | Common | A | |||
48533 | CTT CAT TGT TTT CTG TAG GCT AAA AAG | Wild type Reverse | A | |||
48534 | AAT CGA CCT CCA GCC TCC TC | Mutant Reverse | A | |||
48535 | Fluorophore-1 | TTA AGG GAT GAC AGT CTG TTG ATG | Quencher-1 | WT Probe | ||
48536 | Fluorophore-2 | AGG TCT AAG TTG TGG TCA TAC TAT CTT GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48532 | 0.40 uM |
48533 | 0.40 uM |
48534 | 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 |