Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:55181709-55181797 89bp GAGACCCAGATGTGCCAAAG CGTGCCAGCACTTCAATTAC
Mut= 92 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
ATAGACAAATTATCTAAGTATTTTGGGGCCTCCATTTCCCATCTACAGACCAAACCCCAACCATTGCCTACTTTATTGTCCTGCCAAGACGATTAAATGAGATGTTATATTTAAAGACTCCACGCATACAGGGTCTCATACAATTAGTGTTTCATGGTACAGACAGGGCCCAAGCCGCTAATGGAACAATTCTGGATATCTTGGGATGGGTGGTCACCTTCCAGGAGACCCAGATGTGCCAAAGCAAGGATACAGGAAAAtcgagatggccatgaatttacctgcccacgcaggtaattgaagtgctggcacgctatgaggggatggctgctaggtcctttcccagctccatttccatgtcctctgactgccttcctttcctgtagcgggttccagacattggttcatctggtcaaaggcaacatgggcacagggatcctcggacttcccctggctgtgaagaatgcgggcatcttggtaagaggcatggctaagtgggggcactacgaagaagctgtgggatagggaagaagacataatggtttaaaataccttgtagcagtcagtgcttggggggtggagaggtatctgacccagacataaaatctgaacatcaagtctgagtctgagttctgccttggcttggcctctggcattaggagtcaagtcaacactttttatccaccctcaggcttcaaatccctccttttgtagaatgtaaggattagaggaggaccagTTACCCAGTCTACCTTTACAGATGCCCTTGGGGACATCTCAGAGACCACCAATGGGCCATGTGATCAGGACCCTGAGTCTCCTGCTCCATCACAAATGTCACCGTGCTTGCTATGTGTACTTTTTAGACATGACTTGAGGTTAACTTTCGGAAAGAGTAGCCAAGTCTCTTTCAACTTGGTTTTCTAGAAAGTTCTTTCAGGACTAGCAAATGGCTCACGAGATAAAGGCACTGGCCACCAAGTC
This mutation is a 479 bp deletion beginning at Chromosome 11 negative strand position 55,181,761 bp and ending after 55,181,283 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38476 | GAG ACC CAG ATG TGC CAA AG | Common | A | |||
38477 | CGT GCC AGC ACT TCA ATT AC | Wild type Reverse | A | |||
38478 | CCC ATT GGT GGT CTC TGA | Mutant Reverse | A | |||
38479 | Fluorophore-1 | AAT TTA CCT GCC CAC GCA | Quencher-1 | WT Probe | ||
38480 | Fluorophore-2 | CAG ATG CCC TTG GGG AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38476 | 0.40 uM |
38477 | 0.40 uM |
38478 | 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 |