Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:95790391-95790529 139bp TTAAGGGTTTTTCGGTGCAT AACTGAAAAGCAGCCAGTGC
Mut= 139 bp
Wt= 139 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCATTTTTTAATTGGGTTGTTTGTTTTTTTGGTGGTTAGCTTCTTGTTTTAATTTCTTTAATGGATTTTTTTTTTCTTTAGTGCCAAGTGTCTACAGAATGAGGTAGTCTACTTGGAAGAGTACCTTGAACACAAATTTAACCTCAAATAGTTAAGGGTTTTTCGGTGCATTTAATAAATTATCCATTAGttaatttcttattaccccctccatgtaattagtccatgtaaaagcaattacgtgaggctggagagatgctcaggttggaagcactggctgcttttcagttcctttctggcctccgtaggcactgcatgtgggtgatgtgcatacaagcaggcaaggcacccataagcacaaagtaacagcatggaggtttttaaaagttagctgtttgaaaacagtatatttgggaaaagttaatgacttttttttatctcttctctccagctacatatccccatcgtttgaatctcttttatcttgccaatgatgtcatacagaattgtaaacggaaaaatgcaatcatatttcgtgaatcatttgctgatgtgcttcctgaagcagctgctctagtgaagtaagtaaatctgtattgtacttggggattaattttttaatttttattttcttgtttttgcacttattaacaaaaacataaattgtgcatatttaaagacccaagtaaaatattgtgttattggacaagtgTAATGGTGTTTGTTTTTAATTTCAGCATCTGGGGAGCGGGGAAGGTGAAACTTTTAAATTTGAGACCTCGTTTCAAAAAAGCAAAAATGGGGTGAGCATAGGAGGTAGTAGCTCATTCTTATCTTTCTCTGGATTCATTCATAGTTTAATTTTTAGTGTTC
This mutation is a 522 bp deletion beginning at Chromosome 3 position 95,789,969 bp and ending after 95,790,490 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41629 | TTA AGG GTT TTT CGG TGC AT | Common | A | |||
41630 | AAC TGA AAA GCA GCC AGT GC | Wild type Reverse | A | |||
41631 | TAT GCT CAC CCC ATT TTT GC | Mutant Reverse | A | |||
41632 | Fluorophore-1 | AGC AAT TAC GTG AGG CTG GA | Quencher-1 | WT Probe | ||
41633 | Fluorophore-2 | ATC CAT TAG TAA TGG TGT TTG TTT TTA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41629 | 0.40 uM |
41630 | 0.40 uM |
41631 | 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 |