Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:117549217+117549332 116bp CCTGCCGTCTGCTATCAAG TGAACACAGCACAGCACATC
Mut= 116 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATAGATTTACTTGCCCTTAGGCAGTGTGTGCATGTATAAAATGTATGCAATATATATAAAATATACACAATGTAGACAGCACTGGTCCATATCGTCTCCTTTATGCTTTGATCCTGAGGATTCCAGCTCTACTGTATTCCAACCATTATAGCAGCACCTCCTGCTGCCatttgaacagaaccccagagggaaattttccccagacaggagagtggaggaggggatcttggagaattgcagtcactgtcctggtcactgtgagcgtggagaggctgtcttgtgggagcagagtgggaagctagtactcagagaaaagcatttccacagtccgagccaatgagagagaagaatggcagtcttggctcgggcttctccacctgccaccctctcggggccagcctgcttaccagcatggctcagtgacaattttctgtttctctaatcctccaggatggctacttctctcaccgtcccaaagagaaagttcggaccgacagcaacaatgagaactcagtccccaaggactttgagaatgtggataacagcaacttcgcccccaggacccagaagcagaaacaccagccagagctggcaaagaagccccccagcaggcagaaagagcatttgcagagaaagctggatgcccaggacaaacgacagggccagtcagtcctggggaaaggccccaaggaggtgctgcctcctcgggagaaagccacaggcaacagtagccaagggaaggatctctcaagacacagccatgccaggaagagtggtggtggtgggtcccccgaaaccaagtctgaccaggcccccaagtgtgatatctctggcaaggaggccatctcagcactgacccgcgctaagtccaagcattgtcgccaggagattgcagaaacctactgtcgccacaagctggggctgctgatgccagagaaggtggctcgattctgtcccctagaaggtaggtctctttctcctgtccctggccccatctctctgccttctcaggccctctgaatatagccacactgcacctcaaaccctgaccacaagatctggtttgtctgggaactctgaactcctgccgtctgctatcaagaaagtttgacaaaatcatgacatgtgatagtatcatatgacctgttccctttagcagATATGATCTCGGTGTCTGCAGATGTGCTGTGCTGTGTTCATAGCTATCTGGAATTTACAGGCTACAGAAATTAGAAAAAAATTAGACTCTGTCAGACGTAGGCTTTTTACGTATGCAATATTGTACAGCCCCAACATTACATA
This mutation is a 991 bp deletion beginning at Chromosome 7 position 117,548,302 bp and ending after 117,549,292 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41655 | CCT GCC GTC TGC TAT CAA G | Wild type Forward | A | |||
41656 | TGA ACA CAG CAC AGC ACA TC | Common | A | |||
41657 | CGT CTC CTT TAT GCT TTG ATC C | Mutant Forward | A | |||
41658 | Fluorophore-1 | CAT ATG ACC TGT TCC CTT TAG CA | Quencher-1 | WT Probe | ||
41659 | Fluorophore-2 | CTG CCA TAT GAT CTC GGT GTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41655 | 0.40 uM |
41656 | 0.40 uM |
41657 | 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 |