Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:87224761-87224920 160bp TCAATCCAAACTCAACAAGCA CAGGTCAAGCTGCCTCCTC
Mut= 160 bp
Wt= 160 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTCCAAAATCAAAGCAACAGCAATCACATAAGAAGCCCAGTCTCTGCCTCAGTGGTACCAGCTGCTTTCGCAAAGGGGTTATTGGGGAGTGGGGGTGGGGCTTAAGCTCACAGAGGCTTCCAGGAACCAGCGGCTGGTTTCTGTGGCCACGtgacttaagctgcttcctgaaacacccttaccgccatcctctccccacagctgatgtgcaggtcaaacttctctgcattattgtgttctttctccctcccaggtgagttacggaaactaggggtccggtgcgtctatgacaacgctgctgtggcgagctgggccaaagtgttgttcctctgctgtctgcctgcccagctgcctaatatctgcctagaaattcaatccaaactcaacaagcactgcaccgtgtacagctttgtatccgccattccgctacccaggtatcttgtcaggatgagagcggaactcatagcctcttggcctacctcttggagTTAGGCATGCTCAGTAATGAGGAGGAGGAGGCAGCTTGACCTGCAGAAAGACCCCGCCAGCCTTTCTAGTTCTGTCTTCCTCCAGTTTTACACAAACAGCACTTGGGGTTTGGATGACTTTCCCTGCCTAAAATATTAATGGCCTGCTGCTTTCCAGTACTCCATTCCCACTTGGGGCAT
This mutation is a 337 bp deletion beginning at Chromosome 12 position 87,224,804 bp and ending after 87,225,140 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46894 | TCA ATC CAA ACT CAA CAA GCA | Wild type Forward | A | |||
46895 | CAG GTC AAG CTG CCT CCT C | Common | A | |||
46896 | CCA GTC TCT GCC TCA GTG G | Mutant Forward | A | |||
46897 | Fluorophore-1 | CAG GAT GAG AGC GGA ACT CAT A | Quencher-1 | WT Probe | ||
46898 | Fluorophore-2 | CTG TGG CCA CGT TAG GCA T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46894 | 0.40 uM |
46895 | 0.40 uM |
46896 | 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 |