Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:84473300-84473398 99bp GGCCACTGAAGTCTTTCTAGC CTCCAGTGCCTGTCTCATGT
Mutant= 90 bp
Wild Type = 99 bp
Wt Sequence (deletions in lower case):
AGGAGAGAAAGAACTGAGCTCATTCTTCAGAAGAGGGGAGGGCCTTTCAGGAGGAATTGCTTATCAGACTCAGCACCCTTGAGACCTGAGGGCAGCCTGTAGGATATACCCGAGGAACTTAATATCAGGGGCAGAGTTCATTATGGTAACAGGCAAGGAGTTTGCCAAACTTAATGACTTTAAGAACTGGCCACTGAAGTCTTTCTAGCTGCAGACACATTTCTAAGCACCTTCCACgtgctgctatcttgcttattccttagcgtcacatgagacaggcactggagtagtttccatggagttgatctcagaaagtactgactgtccccactcagaacttgtcaggagacagggagtgacagcaattggagagttgtaaatgactaattatcccttttctctgctttatttctattccaggtcacaaagcacttaaagccttgttaaggtcattggtggatcttcaggaagagttgcttttccagtacccagacacgagacatatagtaaatggggcgaaacccaatacagagaggtaaggaggaagcagagccatggcttgattattttcttctcatctgaatgaaaaaggaaggcgggtctttctcagacagatagatacgggatttgataaacgtaaaatagagtcgcttttatatttaacAGGTGGTCAGAACAGGACAGTTTAGAGTGCTTGTTCTAGTTTTTTTTTGTAACTGCTTATCTTTATAGCTTAAGGCAAAATATTTGCCTTCTCTTTTGTTCTATAATAGTGTACTGGCTAGTTTTGTGTCAACTTGACACAGGCTGGAGTTATCACAGAGAAAGGAGCTTCAGTTGAGGAAATGCCTCCATGGGATCCAACTGTAAGGCATTTTCTCAATTAGTGATCAAGGGGGAAAGGCCCCTTGTGG
A 427 bp deletion beginning at Chromosome 11 position 84,472,923 bp and ending after 84,473,349 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49582 | GGC CAC TGA AGT CTT TCT AGC | Common | A | |||
49583 | CTC CAG TGC CTG TCT CAT GT | Wild type Reverse | A | |||
49584 | AAC TAG AAC AAG CAC TCT AAA CTG TCC | Mutant Reverse | A | |||
49585 | Fluorophore-1 | CAC CTT CCA CAG GTG GTC AG | Quencher-1 | MUT Probe | ||
51370 | Fluorophore-2 | TGC TGC TAT CTT GCT TAT TCC TTA G | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49582 | 0.40 uM |
49583 | 0.40 uM |
49584 | 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 |