Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:78246026+78246110 85bp AGCCTGTCTGGTGTTTAGAGAT GAGTTCCTGTGGCTGGGAAC
Mut= 83 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
AGAAATTAATGGACTTTTAATATTCAACAAGTTTTTTTTCTTGTTGGTCTTCTTACTTGGCTGGCAGGTCTGTTTGGTCTGCTGATAGGTTGGTTGGTTTGAGCAGGGGTGTGTGTAGAGGGTAGGGTAGGGAGTGGACGTGTTTGTATTTGTCTGTGTGTATGAAAAAAAAACTTTGAATAGTTAAAGTAACACATTTGTCCTTTTCAGAGGTGCGGTTACATTAACACAACTGTGTTAGGTAAGCTCTCTGCATCCTTCTAAAATGGAAGTTGCTTGATTTGTCACAAAGATCTCTTGATAGTTTTCATGCTTTCGCTGAGGAAGATGAGTAAGAAAGCCTGTCTGGTGTTTAGAGATTGAGAACCcctctctggtctaaaatggctggtgttctgctcgtgttcccagccacaggaactcatacaagaagagcatctgcgtggacatgctgcgagatgggttccacaagtccttcaccgagctctttgcactgatggaacagtgggataagctccgggaagctgcgaaggcacagtccctcttctggcagcagaggcctctggaggaccagccggacaagctagacaatttctaccactacctgaccagggccgaggctgccgagaggaaaggtaggtgctgggatgtggggttgcaccccagggaagattccaaaatgtcaccctatgaatttgatacattaagacaaatcacacagttcaacctattccactgagtggctttaaggCTAAATGACCAGCTACAAAGACATTCTGCAGATGTCATTGCAAGCCTGGGAAGACAGTGTTCTACCAAACATTTCCATAAGTTTCCAACAGGTGTAAACCTGTCCTATCATTGAGAACACATCCTGATTTGTCTTTGTGTTGTATGAATATTGACCAGTTTTCAAGGCACTGTCACATAAAAACCACACAGACATAGTTTGTTTTAGCATCTATGTAACGAGCGTTCAAAGTTTTGTCATGAGATCCTGTCAGCTTGGATTAGAAAATGGTAGTATTTAAAAAGACATGAAAAGCCTGTAATTTCTAGGGTATTGTTAAGGCTGTAATCCCAGCACTTGGGACAAAGAGACAGCTTATTCTGAGTTTGGGGTTAGCCTCAGCTACAT
This mutation is a 383 bp deletion beginning at Chromosome 8 position 78,246,056 bp and ending after 78,246,438 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48399 | AGC CTG TCT GGT GTT TAG AGA T | Common | A | |||
48400 | GAG TTC CTG TGG CTG GGA AC | Wild type Reverse | A | |||
48401 | CTT CCC AGG CTT GCA ATG A | Mutant Reverse | A | |||
48403 | Fluorophore-1 | ACC CTA AAT GAC CAG CTA CAA AGA | Quencher-1 | MUT Probe | ||
48406 | Fluorophore-2 | CTG GTC TAA AAT GGC TGG TGT T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48399 | 0.40 uM |
48400 | 0.40 uM |
48401 | 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 |