Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:84732093-84732195 103bp GGTACCAACTTGTCTGTGTTTGG AAGTTATGTCAGTAGGAGTGGGACT
Mut= 137 bp
Wt= 103 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TGATTCTGGACGTATAAGCCGAATAAACCTTTCCCTCCTCAACTGACTTTTTGGTTATGGAGTTTTATGGCAGAAATAGAAGCCTAGACTCAGACAGATATATTTTCCTTTTCTTTTAAATTGTGGGTACATCAATCCATGGATTCACGTTGTTTCTCTTTAAATATTTGCTTATAGCTCTTTGAAGATTGTTTTGTTAATTGTAGATTAAATTCAGACTAGACTTAGCTTTAATAGATAttgttaaaggttttggtaattttgttaaaaatgactttgcatgtggatttttaaaaaaaaattcacatatatatgtatacacacacacacacacatatatatgtcataaatacataatatttctgcaaaagtaatccccagaattttgcttcaattgttctgtttcataatgtagtgcttaagcatgtaatctttcattgtggattttttcacatttctatcataaatcaattttccatccctagatctgagtgagtggtactgcagtaagtccatcctggctcttgtggacggacaaccgtgggacatgtataagcctctgaccaagtcttgtgagatcaaatttcttactttcaaagaccctgacccaaaagaagtgaataaggtattttcctgtctcctccatttttttcttcttctaaatcaacctatgtgcaatggagtatatagtctaaaaatacagacaaactcatatgtggtattctactaaatagtgttttactaggtatttttgtagttcaaaatactggtggtaccaacttgtctgtgtttggattgaccgcatgtcatttgtgctcataactttggataaccctctaaATCATCGGGAGTCCCACTCCTACTGACATAACTTGAATTTGTTCTGAAATCACCGTCTTCAGTGGATGGTTTTGGTAACTTTTTGAGTCTGTCCCACCCTCAACCTCCTGACTTTTACTGCTTAAAACACCACCTTTTATAGTTGTATTAAGTTTTATTACTGACGTTACGGGGTGTCTATATCTTTTATCTCTGAAGAGCCTGCCTTCGAGTTAAAGCTGCCATAGCT
This mutation is a 600 bp deletion beginning at Chromosome 16 position 84,732,127 bp and ending after 84,732,726 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64671 | GGT ACC AAC TTG TCT GTG TTT GG | Wild type Forward | A | |||
64672 | AAG TTA TGT CAG TAG GAG TGG GAC T | Common | A | |||
64673 | CAT GGA TTC ACG TTG TTT CTC | Mutant Forward | A | |||
64674 | Fluorophore-1 | ACC GCA TGT CAT TTG TGC | Quencher-1 | WT Probe | ||
64675 | Fluorophore-2 | TTG CTT ATA GCT CTT TGA AGA TTG TTT TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64671 | 0.40 uM |
64672 | 0.40 uM |
64673 | 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 |