Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:64389119-64389220 102bp CAGCCTTTGCTAAGTTGTGC ACATCACCACTACCCCACCA
Mut= 100 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATACTCTATTTTACCTGTAGGTCTCAATCCCATTTCAGATATCATATCCTTAGATAATTTTTCTCAAGCTTAAAGCCAGGTGGGAATCACTTGCCAAACGACTAATAGGTGAGGAGTATGTATGATGCAGATTTGAGGATTTAAGGCTGACTTCCTGGgtatctccaggcaagtagatgcgtaagcttcagtatatacagcaaagtcaggtttttagtttttacggtctggctcatcagcagggtactgttgggaactagagtctgtacttaaaattgatgcttcatttacattttaatttgcaaatacgatatgtctttttcttttatttaaaaatgcttgatgctattgtgtataattcattctcacttaattttctttccaatagaattcagaatggactagcatcaaacttcacatctttaacaaaagttctatatgattttaataaagtattagagaatggccgaatctctggaagtcctttacagaaacttgagataaacagttttgatgatgagcagatttggcaacaactcgaattgcaaaatgaacctgtcttacaatacttccagaatgcagttagtgagacagttgaggatgaagatatcagccttctcccagaatgtgaagatgaggagtgcgaagaggatgcctcagaggtggaggctgacaaccaggagaacctagagacagacttggatgaagaacaactgtcagacgagggtggtgatgttcctaaagggagggacagagccaaaagctccaggaaatctgacccaaggaaaagtccagttttcagtgatgaagattctgaccttgactttgatattggcaaattggagcagcagaccaagatgcaaatcaagccacctggaaaaccaagagaaaagtctgtcgtagatgataagttcttcaaactgtctgaaatggagagcttcttagaaaaagtagaaaaagaagaggaaaaaagaccagatggtgaagaggaggacgaggaagacattgacctttttgaagacattgattcagatgaaagcgagggagggctgtttgggcggcagaagattaaggtaatgttttggaaaaaaacaaaaactgtttctttaaagtacaagtcatgttttcctacagagaaatgtaatgtgcctcagacctcatttgagcccaaatgaagtaaggaaaaaacaaacaaacataaagaaataaattaaaattaagagctgagccagtggtgcaggagtgtagtaatcctcagaagtgggaggctgaggcaggaagattgtggcttctcaaagccagcctttgctaagttgtgcttcagttgtccttatggttttatggtgGTCAACTAGTTGCCAGTTTCCCTAATAACTATTTCTGGTGGGGTAGTGGTGATGTTAGGAACCAGTTTGTGTGTGTAATATACATAGGCACATGAGTATGTTTATATATTTTTTTATCTTTAAACTTTTCAAAAATGTTTTTTATTTTAGTCAAATAAA
This mutation is a 1188 bp deletion beginning at Chromosome 7 position 64,389,174 bp and ending after 64,390,361 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44000 | CAG CCT TTG CTA AGT TGT GC | Wild type Forward | A | |||
44001 | ACA TCA CCA CTA CCC CAC CA | Common | A | |||
44002 | GAG TAT GTA TGA TGC AGA TTT G | Mutant Forward | A | |||
44003 | Fluorophore-1 | CAG TTG TCC TTA TGG TTT TAT GGT G | Quencher-1 | WT Probe | ||
44004 | Fluorophore-2 | AGG CTG ACT TCC TGG GTC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44000 | 0.40 uM |
44001 | 0.40 uM |
44002 | 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 |