Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:63143523-63143642 120bp AGGTAGCTCTCAGGAAGTGAGC GTGGATTTGGGATAATCTTTGG
Mut = 128 bp
Wt = 120 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGGTAGCTCTCAGGAAGTGAGCATAGAACGTgggggtggctttggacagccgctggcttgaaagcggctttgtgttttaagtaatttgttccggagtaccaaagattatcccaaatccactacggtacgtcatcactgcgttcaggtctccagggcccaccaacctctgccacactctcgtccttcctgttggcccgctgggcaagggtgtcccgcctaatcgcctgtcattttcggccgtcgcaggtgtatgcgcctgaaagggacgaacacgtgaagagtacggcggtggcggcggcgctggggcccgcggcctcttacctctgcaccccagagacccacggtaatgcctcctgaggtctctggcccttttaatGCAGGAAGTACAGAGAGAACCCAGAGCCAAGGCAGGCCTTTCAAGGAAGGGAGGGGCGGAAAGAGACGGAGAGAAA
This mutation is a 345 bp deletion beginning at Chromosome 9 position 63,143,267 bp and ending after 63,143,611 bp (GRCm38/mm10). There is a 2 bp insertion (AC) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51574 | AGG TAG CTC TCA GGA AGT GAG C | Common | A | |||
51575 | GTG GAT TTG GGA TAA TCT TTG G | Wild type Reverse | A | |||
51576 | CCC TTT CTC TAT CAC CCC TTT TC | Mutant Reverse | A | |||
51577 | Fluorophore-1 | CTG GCT TGA AAG CGG CTT T | Quencher-1 | WT Probe | ||
51578 | Fluorophore-2 | TAC GCA GGA AGT ACA GAG AGA ACC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51574 | 0.40 uM |
51575 | 0.40 uM |
51576 | 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 |