Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:36357723-36357823 101bp TTTCCCTCTATAGCCTTGTAGTTC CAGAAGGTACCCTTGGTCCTC
Mut= 100 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGTGTTGTAATCCCAGCACTATGGAGGCCGAGGCAGGAGAAGAGTTTAGAGTTTGAGTACAGTTTGAAGTGAGACCGTACTGGAAATAAATAGAACTGAAACTTGTCTTTTGTTACAAATGTGATTATTTTAGCCACCAGATAAATAAATAGCTTTTGAGCACTGAAAAAAAAGCTCCATGAtttgttgtgacttgcggtgtatggtaaggtgattcattggcggaattgggaccaaaagctagagttcatgctttagcttagtggccttagactttttgtaatgagaacaaaaatatgacagtatctctttatatggtggacatacatgggaaatgtcctcagggttgcattttaattcctcagagtttctgttataggactggctggacagtggctcagaataagctatttaacaagatcctcaaagccctgcagtctgaccggcttgcacgattggccaatgaaggggtaagacattggaagtctgtctatagttgactctcttagttttggcctcgggggttgttgtgtcctgtgtgctctctcccctcagcccccagcttgctcacggagcgtaactcttgttttattctttttgtgaacacctttcaggcttgcaatgagcctgtgctgcgtcgcgtggctgtggacaagtgtgcaaggagggtgcggcaggcgctggcaagtgtcagctgggacaccaagctgacccagtggctgcacaccaccctcgtggagaccctgagcctgcccatgctggctgcttacttggatgccctgcagacactgaaagggaaggtaagactgtgcttggtttgcccgagtggcagcagccagggtttgctgtgagttcagagggtttgcacaaggccctgtgactaggtcagcacagaagctaacaatttggctattataatggaaaaggggatcattttcccttctatttggtataaatttcttcagtcgtgccatttttttggataaagtgtggtatctgaaaaccaggatacataatttccctctatagccttgtagttcttaccttgagtcatcgctgacttgaactgtggtggaagagACGAGGCTGAGCAGAGGAGGACCAAGGGTACCTTCTGAGTTAACTCTCCTCATGAAGAGATGGACTGTAGGCTGAGTATGGTGCTACACACCTTTAACCACAGAACTGTACAGGCAGAGGCAGGTGGCTCTCCATCACTTGGAGCTTGGTCTACACTGCAAGT
This mutation is an 898 bp deletion beginning at Chromosome 1 position 36,357,760 bp and ending after 36,358,657 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43995 | TTT CCC TCT ATA GCC TTG TAG TTC | Wild type Forward | A | |||
43996 | CAG AAG GTA CCC TTG GTC CTC | Common | A | |||
43997 | TGT GAT TAT TTT AGC CAC CAG A | Mutant Forward | A | |||
43998 | Fluorophore-1 | CTT GAG TCA TCG CTG ACT TGA A | Quencher-1 | WT Probe | ||
43999 | Fluorophore-2 | AAA GCT CCA TGA ACG AGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43995 | 0.40 uM |
43996 | 0.40 uM |
43997 | 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 |