Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:84159153+84159265 113bp TCATGCCTGTATTTTGGAAGC CCCATTTCCTTATGTGGCTATAC
Mut= 100 bp
Wt= 113 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTCTTGTTGAATTCCAGGGCCCAGTGAGAATCCTTGTCTCAAACAATAAGCTGAATGACCCCCTAAATATACTAATATGTTCACACAAAGTTCTTGAATCAGCCTCCAAAATGCAGGTCACCTGCTCATGCCTGTATTTTGGAAGCTGATGGCGGCAGGAAGTTCATAgccacagggtaagatcccatctcaatgaaaaagaaaaaaatctaattgtatagccacataaggaaatgggccctgcatcttgaaatgggctccttctcatcttcccatcactaggtgagcatcagtactggccgccttgccttcatcaaagacccactcacctagtccagctgtggtcctgttgtgctaagctaccagctatactaaacctgactacatccctgccccttacttccctctctaaatcaggggtcggcctccatccctcaaagccttctaagaacccacggctgagagactcccagagctccatgatggtccattcacactactccagagagtcggaagacagctcagaacctgctccttctgtagagctaggcggggaggaaccactacacgaagctttcaggtaggtggctcagaactctcatgcaggttcctccctcttcagtcattcacagcaagcccataggaaagacaaatgatccaggatgacctgccaccactgcaaccctttctgggaccctgGCCCAGGTCTTGTTTCTCATGGTTCTCTTTACTGGAGAAAGGTCACAGATAACATCAATTCACTCTTAGTCTCAACCACACTACTGGAAGAAGTTGGGAAGATAGAGCAAGTGCTGAGGAACCTGTTTCACAAGCACTAAGACCTGAGTTCCATCTCCAGAACCCATGT
This mutation is a 531 bp deletion beginning at Chromosome 8 position 84,159,196 bp and ending after 84,159,726 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44873 | TCA TGC CTG TAT TTT GGA AGC | Common | A | |||
44874 | CCC ATT TCC TTA TGT GGC TAT AC | Wild type Reverse | A | |||
44875 | TGA TGT TAT CTG TGA CCT TTC TCC | Mutant Reverse | A | |||
44876 | Fluorophore-1 | CCA CAG GGT AAG ATC CCA TCT | Quencher-1 | WT Probe | ||
44877 | Fluorophore-2 | AAG TTC ATA GCC CAG GTC TTG TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44873 | 0.40 uM |
44874 | 0.40 uM |
44875 | 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 |