Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:45113088-45113234 147bp CCATCATTTTTGTGGCAGTG TCACACATAGGAAGCGCAGA
Mut= 154 bp
Wt= 147 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCTGTCCTGCCATTCCTATTGCTCCTTCATATATTTTTCTTTTATACACACAAATGTTACAGGTGGGAAATACATGTTCTTGTCACCAGAAAATATGTCACAAAAAATTAACTGATCTAAAGTTGCCTGCTAATGTATAAAACCTATTTGCATAAATTTCTAGTAAGAAAAAAGTGTCTTTTTAAAAGTCATAAGTAGGAAatctgtcagggaccaccatggacaaatatgaatatatattcctatattttgcacatatgaaagtgtgacctctctgtcaaatttaattctcttgtacgtctgtgttttttttttttttaattaatcacaaagttgtcttctttgaaagatacttcagaagaaaggagctgagtgaaactcttgactttaagaaggacacagatgaccaggaaagtcaacatgagagtgaacaggacttctcaaaaggaccttgtcccactgtccgacaaaagctctgggatattctggagaaacctggatcttccacagcagcccggatctttggagtaatctccatcatttttgtggcagtgtccatcgtcaacatggccctgatgtcagctgagcttagctggctcaacctacagctgctggagatcttggagtatgtgtgcatcagctggttcaccggggagttcgttctgcgcttcctatgtgtgaaagacaggtgccacttcctgaggaaggttccaaacatcatagacctccttgccatcttgcccttctatataactcttctggtggaaagcctgagcggcagccacaccacacaagagctggaaaatgtgggacgtctggtccaggtcttgaggctcctcagagctctgcgcatgctgaaactgggaaggcattctacaggtattcatatttcactcgtgccttttaatttcgcttttaaaataatctttagatatattggcacagaaccaccttacattcttactcaccctccaTCTATAAAGTACTAGGAAGAAAAAACTTCAAGATGCTATCATAAGCAAAGATATCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCATATATATACACACATACTCTTTCTGTTCACAAGACAATTTGGACATAACAATTTGATG
This mutation is a 773 bp deletion beginning at Chromosome 15 position 45,112,795 bp and ending after 45,113,567 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39053 | CCA TCA TTT TTG TGG CAG TG | Wild type Forward | A | |||
39054 | TCA CAC ATA GGA AGC GCA GA | Wild type Reverse | A | |||
39055 | CTG ATC TAA AGT TGC CTG CTA ATG | Mutant Forward | A | |||
39056 | GAG AGA GGA GAT ATC TTT GCT TAT G | Mutant Reverse | A | |||
39057 | Fluorophore-1 | CAT CGT CAA CAT GGC CCT | Quencher-1 | WT Probe | ||
39058 | Fluorophore-2 | AGG AAG AAA AAA CTT CAA GAT GCT AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39053 | 0.40 uM |
39054 | 0.40 uM |
39055 | 0.40 uM |
39056 | 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 |