Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:21893103+21893270 168bp AGGGTTGGAAAGCTTCAAGG GGAAACAAGGCACTCTCCAG
Mut= 170 bp
Wt= 168 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
GCTCATTGAGTGAGAGCATTTGCTGGTTTTGGAGAGGACTAGTTTAGTTCCCAAGACCCATGTCAGATGGCTTGAAAGTGCCTGTAGCTCCAGCTCCATGGGATCTAGCATTCTTTTCTGGCCTCCACAGGCACTTGCACACACTCACACACTCCACAGAGTGACAGATTTTACATATTCACTACTTAATTTTGATAATTTTAATTTTTAGATGGAAAAAAGTAAAACCTTTATTTTATTTTTTAATTAAATTCCAAATTAATGCctttgttatcccaaagaaactcctttgcttatcatttgcaatacaccatgagaatatcataaaaattcttcaactgtgttggtatcaggactcacatgctcctctctgcttttaacctttgttttcagcttactcagggacgcagctcactaggaatctacagattaagccgtgggaactcggtgaacacgggaccatgtgctttagatcctacaggatggctctctcctgtttgagcagagtaaaaacttacaggtgagtctgtcaattattctagtagagatttacatattttgtgtaacagcagtttagttttgattttgtttaataacctttgccagtatgttaaagcttatgtggtctaatttaagtcgcttttacttgtttaaataattttaaataattattttttaataatttttagggttggaaagcttcaaggtcacctaaaagttagaagggaaatatagtacattagccatgaatggtatttatttacgttcagagacaattattatttttaatgaaaattagacacatttctttcaggttcaccagcCTCCTTCCACCCTGGAGAGTGCCTTGTTTCCCTTAATAACCCTTATTACTAGTGTTCTATTGCTGTGAAGAGACACCATTACCATGGCAAGTCTTATAAAGGACACCAGTTAACTGGGGCTGGCTGACAGGTTCAGAGGTTTAGTTCATTGTCACCATGGTGGAAAGCATGGCAGCATGCAGGCAACATGGTACTGGGAAAGGAGCTGAGAGGTCTACATCTGGATAGG
This mutation is a 559 bp deletion beginning at Chromosome 4 positive strand position 21,892,681 bp and ending after 21,893,239 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38424 | AGG GTT GGA AAG CTT CAA GG | Wild type Forward | A | |||
38425 | GGA AAC AAG GCA CTC TCC AG | Common | A | |||
38426 | ACA GGC ACT TGC ACA CAC TC | Mutant Forward | A | |||
38427 | Fluorophore-1 | AGA AGG GAA ATA TAG TAC ATT AGC CAT G | Quencher-1 | WT Probe | ||
38428 | Fluorophore-2 | CAC TCC ACA GAG TGA CAG ATT TTA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38424 | 0.40 uM |
38425 | 0.40 uM |
38426 | 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 |