Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:58363982-58364075 94bp GAGCCCACCCCACATTAACT CACCTTTCACCCATCTCAGC
Mut= 92 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAATTCAGTCTTCAACATGAATCCTTCCAACTCAGGGGGTGCCAGAATTGTCCCCACTCTGCAGATGAGGAAACTGAGGCTTAGGGAAGGGAGCGCAACATTGCTCTGCAGGTATTCGGTTAAGTGGTGGCTCCAGAGTTCGGGGGCTACGTGGGGGGCGGGGACACTGAGCCCACCCCACATTAACTTGTCCCCACACTGTGCCAGCCAcatgttgggaacgcttgctcaaggccactcaggctgagatgggtgaaaggtggggaccaggtgggcaaggctaccccattccccttgaactcagcatcagctgctcccgaactatcccttcctggaaggggaaggtcaccttactcagccccaatgctgttctcaggaggaactataactccttgtgccagatgtatgagaaaatccagcccctgctggaaaacttacatgggaacttcacagagactcggaacaacatcggtgagtagagggagggagggagagaggaaggaagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggaggaaggaagggctgggcaggggagagggagggagggagggctgggcaggggagagggcaggagccatttatgtcttttcctagtcatttccaggcaTAGGTGACTCCTGGGGCTTAGCAACTGCCTTGATTTTGGCCTGGGGTTTTCCTAGCCAAACCTGGGTCAATCTACCCGCCAGTGCTCATGAGTCCTACGTGTGCTTCCCTGCCACATGCTATCTCTGTTGCTTTGTCTTCTTCTCCCCACCCCTACGACTTTGAACTAAGTCAGATCTGAATTGGATGGGAGGAGCATTTGGGGGCGAGGAGAAGCCCAGAACAGCAGGGTGTGAT
This mutation is a 519 bp deletion beginning at Chromosome 9 position 58,363,515 bp and ending after 58,364,033 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48206 | GAG CCC ACC CCA CAT TAA CT | Common | A | |||
48207 | CAC CTT TCA CCC ATC TCA GC | Wild type Reverse | A | |||
48208 | AAA ACC CCA GGC CAA AAT C | Mutant Reverse | A | |||
48209 | Fluorophore-1 | AAC GCT TGC TCA AGG CC | Quencher-1 | WT Probe | ||
48210 | Fluorophore-2 | CCA GCC ATA GGT GAC TCC TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48206 | 0.40 uM |
48207 | 0.40 uM |
48208 | 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 |