Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:61361406+61361502 97bp CCATCTCTCCAGCCCTAACA GGAACCCCACCAGACAGAAT
Mut= 110 bp
Wt= 97 bp
Fam=Mut
Hex=Wt
WT Sequence: (deleted region in lower case)
AGATCAGAAGGTAACAATTGCTCCATTGATTGTCACAGACACAGAGCCCCGTGGACAGAACCCCATTAGACTATGCCTGCCCATAAAGGACCCAGTGACTTCTCCCTGGTATATGTGTTAAAGATTACCAGCCTTGAGAGTCCAAAATTCCAAAAACTGGCCAAGTGAAGAAGAGGAAAAGCAAAGGAAGCCACAGTGGCCTTGCGACTATGGCTTGCTATGGTTTGGGCTCTGGGAAGACCAGGCATCAAAAGGAACAACAGCAGGAGGAATGGCTTGCCATACAGCGGTACAGCGAAGTATAATGTATTTAAGGTCCAGGTTACAAAAGCCTAGCAGAAAATCAATTCTAGCCAGCAATGCGACATTTTAGCAGTTCCTTTGGAAGGTGTCTCCATTCTTGTCCAAATCTGTACTTCTTATTTTTAATTACTATTTATCTTGTGTGTATGAACGCTTTGCCTACATGTCTCTGTGCACCATGTGTGTGCCTGATGCCATCAGGAGCCAGAAGAAGGTGTTGAATCATGTGGTCCTAGGGTTATGGGTGGGTGGGAGCTGCCAGGTGGGTGCTAGGAATCACACCTGGGTCCTCTCGAAAGAGCAACCAGTACTCTTGACCACTGAGCCATCTCTCCAACCCCAGTTCTGTACTTTCAATGTGTTATTAATTTTAAACTTCTACACTGTCATTTAATTTTTTCCCCTTCCTGTCAAGGTTTACCTGTTTGCCAAAAAGAACGATGCGGTTTTAAAATAAACTTTTTTTTTAAAAAAGGTTCATTTATTTATTTTATGTATATGAGTACACTGTAGCTATCCACACCAGAAGATGACATCGGATCCCATTACAGATAATTGCAAGCCACCATGTGGTTGCTGGGAACTGAACTCAGGACCTCTGGAAAAGCAGCCAGTGCTCTTAACCGCTGTGCCATCTCTCCAGCCCTAACATAAATTTTTAAAAAGGGTGTTTCATGTGAAGTAACctgctggttctgttgtgcatggattctgtctggtggggttcctcattctcctttccttatacctttcataaagatataaataaatcatatctgtgtcccctccccatctttttttcccctgtattagagtatgaatcctccacttcccccaccccctctcctggcggccaccgtactcgcctccaccccgtcaggcgctactgctgctgttgctgctgctgctgccgccgccgctgctggaatgggacccaggcctgtgatggggtcctctgaacagattgcacatttacggcctcagactcgtcccagtgtgtaagtgagggtgtctttattaccacgtaaacactcatctcaaacagacatcaagaagcacaagtctgatgggagctactgacaggcttagggctcaaccaagtggcttgtattctgaaaacttCTACCTGGTTATGCATATAATTAGTAAGACACTTAGAATGAGCCTAATGTGAGCCTGGTGGGTGGCTGTCCCGCTGAGAAAAGGCCTTTCGCAGTTTAGAGGCATCTCTGTTCTCTCCTTTATAGGTTGCCTACATAGAGAACTGCTGTCCTTTCATACTGCTCTGTTGTAACCGTTTTATCTTCAGTTTCATTCCTTGTATCAAGATCTTAAGCAGCAGCAGTTCTCAACCTGTGGGTAGTACGCAACCCCTTTGGGGAGGTTGAATGACTCTTTCCCAGGGAGCGTATATTAGATTATTTACGTTACGATTCATAGCAGTAGCAAGATGACAGTAATAAAATATTTTTATGGTGGGGGGCCACTACATCAGGAGCTACATTAAATGGTTGTAACATTAGCAAGGTTGAGAACTGCTCATCTTAAAAACCAGGAAGGTATTCCACACACATCAATCAGGTCTCCCTGCAGCAAGCATCTAGAGAGCTTTAAGTCCCCATCGGTAAGGAATCCAGAGAGGTCCAGTTACCCTGTGCAGTTTGCCGCCAACAAAAGGCTTCTGCTCTCTGTGCCTCTGCACCTGTAGGGTTCAGAGCCCTGGCCGATGCAGGTCGGTCGCCTGCCTGGTCTCTCTGTGTTGACAGACACCTTGTACTCGGCAGTCCTTTTGTGCTCTCTACAAACCAGAGTTCCTTTGATTTTCTGTATCTCAGTCAGCTTGGTGACTGAGCTCAGAAGTGCAGGTGCAGCCTAAGTCT
This mutation is a 436 bp deletion beginning at Chromosome 8 negative strand position 61,361,896 bp and ending after 61,361,461 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37882 | CCA TCT CTC CAG CCC TAA CA | Common | A | |||
37883 | GGA ACC CCA CCA GAC AGA AT | Wild type Reverse | A | |||
37884 | GGC TCA CAT TAG GCT CAT TC | Mutant Reverse | A | |||
37885 | Fluorophore-1 | TGC TGG TTC TGT TGT GCA T | Quencher-1 | WT Probe | ||
37886 | Fluorophore-2 | CTG GTT ATG CAT ATA ATT AGT AAG ACA CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37882 | 0.40 uM |
37883 | 0.40 uM |
37884 | 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 |