Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:116088172+116088261 90bp ACGACACAGGTGACACAAGG GGAAGGAACATACCATATTAGTGC
Mut= 87 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case and insertion with carrots ^g^ (GCACAAATCTAGGTTT insertion):
CTGCCTTCTGCTTCCCAAGCGGTGCACACTTTTAATCCCAGTGGTGGAGAGGAACAGATCCCTTCAGCTTCTGGCCTGGTAGCCTAGCATATTTGCCAAGTCTCAATCCAGTAAGAACCTCTTACCAAAAAAGGTGGACAGCACCTAAGGAGCTATGCTGGACGACACAGGTGACACAAGGTATCACTTGTCAG^acaagggacactactgtagttaccctacagaaagcactaatatggtatgttccttcctgtttatatcccttgcttatctgtaaaatggggccaccagtaatgcatacctcaaaggtgtgaacactaaataaatttgcagatttaaactaaagcctatctcagtgagtactcagaaatgtttgctacaggaaagaaacttagagggcatttgacaaatttctggtgttttctttgctctttagagtgtgacttgctggcgagccaagtttatggaggcctttttttcccatgttctacgtggaaccattgatgtgtcttctgataaacgtctttgtgatcagcgcttctcacctctcctgcacagctcccgtcatgtccgacagctaaccatctgcaacatgctgcagggagcaactgagctagtggcagagcccaaccgtagggttctagagaccctggccagttctctgcataccctcaagttccgccacctgctgttctctgatgtggctgctcagcagtcacttcgacagctgctgcatcagctcatccaccatggggctgttagccaagtgtcgctgtactcctggcctgtgcccgagtcagcccttttcatccttattcttaccatgagtgctggcttttggcaaccaggcccaggtagcttgccttgccgcctctgtggtgaagcctcccgaggccgggccccatctagagatgaagggtccttattgctaggctctcgcaggcctcggcgtgatgcagcagagcgatgtgctgctgcactaatggccacccggcggaagagtgaagtcaagcagatgcccagggctgtacctcccactcgtgtaacacgccgcagcacacaagagagcctggcaataggtggaacagactctaagttgtacctcccagccacctcttatgaggcttctggcacaaaacagccatctgctccagctgcagcctctgcctcctcttccacatcatccaaacgggctccagctagctcagcctcacagcctaagcccttaaagcgtttcaaacgagctgcagggaagaaagggccccgcacccgtcaggggtctggtgcagagtctgaagacctgtatgactttgtttttattgtggctggtgagaaggaggatggtgaagagatggagattggggaagtagcttgtggagctctggatgggtcagatcccagctgtttggggctcccagcactagaggcatcccagcgattccgcagcatttccaccttggagctattcacagttcctctctccacggaggcagctctgacactgtgccatcttctgagttcctgggtatcactggagagccttacactttcctacaatggtgagcacctgaaggaacagggctgggcttgcatctctcaaaagcactgtgcctaatcaag^AGTGGAGAGTATGATTAAGGAGGTGGAAATGTGGTTATTTGTGTCTCGTGGAGGCAAAAGAGAAATACAAAAGACACACAGTGTTAGGAAAGGAAAATGAGGTCTGTAGTCTGGATTTACTATTGGAGgaaaggccaagGGGCTGAGAAGTCAGATAGATATAGAGGGCAATCCAGACACAGACAGGGCTCTGAATTTGTATCGCATCAGTCAAATTAAAGCAACTGACCAGGGATTTTTTTAAAG
This mutation is a 1426 bp deletion beginning at Chromosome 4 position 116,088,205 bp and ending after 116,089,630 bp (GRCm38/mm10). In addition, there is an 11bp deletion (CTTGGCCTTTC) 128 bp before the exon deletion, and a 16 bp insertion (AAACCTAGATTTGTGC) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40844 | ACG ACA CAG GTG ACA CAA GG | Common | A | |||
40845 | GGA AGG AAC ATA CCA TAT TAG TGC | Wild type Reverse | A | |||
40846 | ATA ACC ACA TTT CCA CCT CCT | Mutant Reverse | A | |||
40847 | Fluorophore-1 | AAG GGA CAC TAC TGT AGT TAC CCT ACA | Quencher-1 | WT Probe | ||
40848 | Fluorophore-2 | CAC AAA TCT AGG TTT AGT GGA GAG TAT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40844 | 0.40 uM |
40845 | 0.40 uM |
40846 | 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 |