Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:36450992-36451077 86bp GAGTAAATCAGCGCACCCTTC CCCAATTTGAAAATAAACCATTC
Mut= 88 bp
Wt= 86 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCTGACTCCCAGTTGTTAGGATTATAGGTGGGAGACACCAGGTCTGGCTCTACAGCCAGGATTTTGAAATAACGTGGGTGTTTTGGATTCTAGTTATGCAATGCAGATGTAGTACGTTGTTTCCTGCTTTGGGTCTCTGGGTTTATCTCCTGAGTATtttatttatcccttttattactctctggctacttgataaatatggctttttttttttttaatgcacatttgtttttgtgttcatattggtcatgcggtctatggatagggtcaatgaatagggtacatttagaatttgcatagtgtgatcattctgttgacttttatctatgcttatcttggtaaatcttacagctttgtcaagttagcatgatctgttttaacaaatctaaatttgacttgaaagtttaatttcctaaatcaagcacttgtttttctcaggccttcacagagcttcaagccaaagttatcgatactcaacagaaggtgaagcttgcagacatacagattgaacagctgaacagaacgaaaaagcacgcacaccttacggacacggagatcatgacgctggtagatgagactaacatgtacgaaggtgtaggaagaatgtgagtaaatcagcgcacccttcacggggctctcttaggagaggcTTCAGGGGAATTAAATGGCAGAATGGTTTATTTTCAAATTGGGTTACTTAAAAAAGAAAACTTGGGTTAAAATAGAGAAATAACTTGCAAAAGCTATCTTTAAAAAAAAAAAAGGCATAAAACCCCAGGTGTGTGTTTTAAAATTTTATGTTTTTCACTCTCTCT
This mutation is a 493 bp deletion beginning at Chromosome 18 position 36,451,035 bp and ending after 36,451,527 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42302 | GAG TAA ATC AGC GCA CCC TTC | Wild type Forward | A | |||
42303 | CCC AAT TTG AAA ATA AAC CAT TC | Common | A | |||
42304 | TAC GTT GTT TCC TGC TTT GG | Mutant Forward | A | |||
42305 | Fluorophore-1 | ACG GGG CTC TCT TAG GAG AG | Quencher-1 | WT Probe | ||
42306 | Fluorophore-2 | CTG AGT ATT TCA GGG GAA TTA AAT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42302 | 0.40 uM |
42303 | 0.40 uM |
42304 | 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 |