Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:19235367+19235516 150bp ACAGAGCAGTGACCACATCC GCTGCCTTTTTGCTTTCA
Mut= 147 bp
Wt= 150 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TTCAAGGCCAATCTGGACAACACTGTGGAATCTGAGCCCCCTTCCCTCCCACGATGTTAATGCATCAAATAAAAATAATTAAAGATGCAAAATATAAGAGGTATATGTGACCCATATCAGGAAAACAATCAATAATCAATTAGAGGAAATAAAATAGGAAGCTAAAAAAATTCAGTTACCAAAAAGAGAAGACAGAAAAAAGAGAGAGTAGAGACAGATAGCAAGATGGTCAATAGATATCAACCATATTAATAATCATACTAGATTATTGGTTTCCTTTTTAAACAGGAACTTTGCAGCATACTTACAGAGCAGTGACCACATCCTTTTGTAAGACTGCAGCGTCTTTAAATTTTAAGccactgttaattttgatgtgacagttacatgaacctaattacttttcctttttctccttcagtttgacccaatcacaattgaaagcaaaaaggcagccactgtggtgctgatgctcaagtctccagaggaagacatcttagccaaagcctgcgaagccatctataagtttgctctgaaaggtttgttcttttggtgttaaactttctatctcgtttctgtatagtactatatatatataataatatacataccaggtgctctgataagaataaatgccacagaagcaaaaagagcagttagataaacttgcattggatcatacccaaagtataagacaaatatccatgtgtacaatgttgatataagagttaaactgaaggcggccccaccccttcctccctgctcCCTTCCCCACTGGCCcctttcACTCAGGGTATTCTCCATTTCCACTTTGTTCTTCTACCCCCTCCTTTTCCCTCCCAGCCTCCTTTGCCCAGGCTCATTGGCCCTTCCTAATTTCCTCGGCTTTACCGCCCATTTTCATGCTAATTTTGTAATTTCATCTTCCTTTACAGCTGAATAAAATTCCATTCAAATAAAATTTAAATGATCAACAGGGACAGATAGTTGTATTCAATTAAAAACCAAAACCAAAACCAAACTGTATGCTGCCTTGAAGAAAGATCTTTAGAATATTAAGGCGCAAATAAAAGGTAAAAAGATATCTCATGAAAGGACTAATCAATATAAACATGGAGTGATTATATTAA
This mutation is a 406 bp deletion beginning at Chromosome 2 position 19,235,420 bp and ending after 19,235,825 bp (GRCm38/mm10). In addition, there is a 6 bp deletion (CCTTTC) 15 bp downstream the 406 bp deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48856 | ACA GAG CAG TGA CCA CAT CC | Common | A | |||
48857 | GCT GCC TTT TTG CTT TCA | Wild type Reverse | A | |||
48858 | CAA TGA GCC TGG GCA AAG | Mutant Reverse | A | |||
48859 | Fluorophore-1 | CTC CTT CAG TTT GAC CCA ATC A | Quencher-1 | WT Probe | ||
48860 | Fluorophore-2 | ATT TTA AGC CTT CCC CAC TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48856 | 0.40 uM |
48857 | 0.40 uM |
48858 | 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 |