Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:37882850-37882964 115bp GGCATGTGATAGCTTTTGAGTC CATGGGACCAAGTGCCTCT
Mut= 98 bp
Wt= 115 bp
Fam=Mut
Hex=Wt
The WT Reverse primer (primer 64370) anneals over the nucleotide sequence containing mouse genomic variations rs585271614.
Wt Sequence (deletions in lower case):
AACAAGAAAAAATTTCAATTTCAAGAAAAAAATTTAAATTTAAAAAAAGGATAGAGCCCTTTCTTAGCTTCTGCGAAGGCAGGAATCGCCTTTGGAATAGTATTTCATCTTCTTTCCCTATGGGTATGAAACATTTTGCAATGTGCAGCCTAGAGTAGGCATGTGATAGCTTTTGAGTCAGTAAGTTTAAAAGATTGTAACTTATCCTGATTgtcccctgtgtgccatataactgtgtataaagaaagtgctcagaggcacttggtcccatgggtagaaccacctgccaacaaaggtaaagaccagagttcaatccctgggaacccacatacaccccccccccccccacacacacacaaaagattagagatgtaactttttaaagtggtccctggttacgttactgtagcttgtgttttgcagatggaaaatatcacaagcaaattaaaatagaagagaatgcaactggcttcagttacgagtcactctttagagaatacctacacgagacagttacagaagtttggatagaagacccttatattagacaaactcatcaggtaagtttactaagatagaatgaaatgtgactttaaatgtacagtcagctacctgtgtttatgggtttcctacccacggattcagtggaggatcagggatataggtaaaaattccatctgtactgaacatatacaaacataacaagacaagggtaacgaatggtgtttacattgtgttaggtatcatgtagtctagaggcgatttaaagggcccaggaggaggtatgtgggttttgggatgatactatattgcttaaaataagggatggaaccatgtgtggatatcttagtatctggctaagggagggtatcctagaatcagttctcttaaatagttttgttagaccttggagtttccttcaggatacagtgcttataaagtacagtgttttattttgctccctgtgcacagtgcccagtaatttgctgtagaagcttaaatttaaaacttccatgtagcttgctatatttgggtccatcatgtgtttccctgtggaacagcgcaccatctttactcaaattattttaatccacctctttggtggtgagccctgaagaagcaccaccagatgggggagctcctccaccagggaggcccccaggcattcctccaggcgtgccaacagctctgctacagcttggtaacgatgggactgcaggcgttcagattcttccttctccgcagcctggttcggatccggctggctgaggattatagactcagttcttgccggagcactgtgaagctaggactaatccaccctaatgcctttcagctctataacttccttcggttttgtgagatgctgattaagaagccgtgtaaagtaagaaccattcaccttctcacctctgggtatgaagtaggtatctgaaatgcctcattctttcttaacttactgtgtccgtcatatgtgacctgatcctacactgtgtgccaagcatagactgtcttaagaaaaaaagctgttgatttgagagtgttgacctcgttcgcattcagccattttgctgagctctttgaatttgagtttgtcagggtggtctcagtggccctgcagtctgcaccagcgactcttctgttctttactccatgtgctgggtttcgactgtggctgtgacttccttgatttagacttgagtagaagtgtatctgaagaattatactttccagggttagtctaagcagtaggttgtctcacacggataactgagcatccttgctaggatacgtttaagaagctgagcatggtgccatgtgcctgtgctcttagcaacagggaggctgacgtaggagggagttgtgtgatagtctcaattgtttaattgtttatataaaaacaatataaaaagaaaagaattacccaaccaacagttgcttctttgagttagcaatattgaaattgatataaatcttttcagtttaatctctctgtagatttatttatttattgtatagctcatggatagttaataacagaggcagtaaattatgttttcatgctatcaatagggtttggggaacacacagcaaagtagtggcctggaagaaataaagcagtccctcgggagtcacggagtggtgctggaaataaactactcttcatctatacatgaccgagaaattaggttagtgtataatgttttactttttttgtagctgagatatactttaaaaattctaatgtaatataattaaatacatgtatttaaatattcaaatcaagctcaaatttagtaattgtatattggacaaaaacaaacaaacaaaaaaaacccatcttggtttacctcttagagtgtgttaattcttagtcaagtaatatttacaggtcctatgtcatGTGGGAAGGAGTTCTGAGTTCTGTGCAGACCATAGCCACTGTTATGTTAGGAGATAATTCCTTTCCTACTGAAATTAGTTTACCTGGTGTGGCTTTGGTGGTATGTTCTAGTAGTGTACTGTCGATGAGAAAATGCATGCACAGTTTCTTAGTGATCCTTTAGCATAGTAGATTTCATATGCTACTGCAAAGAGACCCTGTTTCTAATAAAAAGTAAATCAGAAGCTAAATATGTAAAGTGG
This mutation is a 2192 bp deletion beginning at Chromosome 1 position 37,880,718 bp and ending after 37,882,909 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64369 | GGC ATG TGA TAG CTT TTG AGT C | Common | A | |||
64370 | CAT GGG ACC AAG TGC CTC T | Wild type Reverse | A | |||
64371 | AAC AGT GGC TAT GGT CTG CAC | Mutant Reverse | A | |||
64372 | Fluorophore-1 | CCC TGT GTG CCA TAT AAC TGT GTA | Quencher-1 | WT Probe | ||
64373 | Fluorophore-2 | CTT ATC CTG ATT GTG GGA AGG AGT TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64369 | 0.40 uM |
64370 | 0.40 uM |
64371 | 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 |