Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:46788654-46788765 112bp GTGGAAAATGCTGATTTGAGG GAACCGTGAGACACATCC
Mut= 107 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TAAACACTTGTAAGTGAGCAAATGATTGAAGTAGCTGTTTTGTCCCTGGTTTCCTCCACTGTAACTAGCACTGGAATAACCTGGTCAAGTTATGAAAATATGAACTGTTTAATAACTGGTTTTAACAAAGAAAAATTACACATTTATACAATTGAGCCTATTCAGGTTTTGGAGATTCAGCTAAAAGTTTAAAAGGGAAATGTTCTTTGTCTGGCATATTTCAGTATTCTAGGATTATAAAGGAGTGCTGTTACATTGTGTCccgggcaggggagagaaggaaggaagtgagcacagacctgcaatacttagttcaagtggctaggttatttttaagtgttgggaatgcagcctaggttctcgtgcatgccaggtaggtgtcctgtaaccgaggtgcatccccagtgcatagcagatctcacgattgtctcctcatgctcttagtgtaccagcatagctgtgtctcggaaatggttggcgttaggcagcacaggaggaggactcaatctcattcagaaagatggctggaagcaaaggctgtttctctctcaccgggtaagaccaaggacattcacccactcccccccaatgtaatcaattatttagagtaaaagaagaatgaaaacgaactattcttgtctgatgttctaggaaggggcaatctctcagattgcctgctgctctcatgatgatgattatgttgctgtagctaccaggtaagaaacccttaaagctgacatgtgtgcttgtcaggatgaccaaccatctcatctcacccagcactgtcctggtgtcagttctgaaagtcacatcctggggatccaagtctgctcagtgcccaagtcacctgggaggggtcgttggtcaccctggtgtttgtggaaaatgctgatttgaggcaacaacatctaaaaggtcttaagagagaaatggaagagtcgtcacctctcccTGAGGAGCTGGTACACGTCTGGATGTGTCTCACGGTTCTCTCAGGGCCTGCTTCTTTTCTCAATCTTCATGCTTAAGCCTGTGCTATCTCTAATCTTGCTGAGAGAGAGTGGGAGCAGAGGATGTGCAAAACATTTGAACTCTTAAGTTCTCCTATTTTATTGGTTTGATGACCAAAACAAGGAAGTGAATACTTTGACTTGTAACTCCTTCCCCTAAGAACAGGAGTTTATATTGTATTTAATATGGCTAATATTT
This mutation is a 690 bp deletion beginning at Chromosome 7 position 46,788,692 bp and ending after 46,789,381 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48001 | GTG GAA AAT GCT GAT TTG AGG | Wild type Forward | A | Wt F | ||
48002 | GAA CCG TGA GAC ACA TCC | Common | A | Common | ||
48003 | AGG GAA ATG TTC TTT GTC TGG | Mutant Forward | A | Mut F | ||
48004 | Fluorophore-1 | AGA GAA ATG GAA GAG TCG TCA CC | Quencher-1 | WT Probe | ||
48005 | Fluorophore-2 | AGT GCT GTT ACA TTG TGT CTG AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48001 | 0.40 uM |
48002 | 0.40 uM |
48003 | 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 |