Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 120 bp
Wild Type = 121 bp
>chr6:82908212-82908332 121bp GCCTGCTTCCTTCCATCTC GCATCCCTAGAATAGCCCTAGA |
Wt Sequence (deletions in lower case):
GCCTGCTTCCTTCCATCTCTTTGGCTGCAGCACATCTTCTAGGCAGTTACAGACtccagaatttgagaggaaaggtctttgtgcagcctcatggtattttctagggctattctagggatgctttcatgggggaagtgtgggggacaggtctgggcggcaaagaggaataaagcagagaaagaaatgagtgggtggaggaggcaggggtaaggcagctagctgaatacggagatgggagtggggtgagcggggtacgaatggagatgggcttttccttcatttgatcttatgtttaattctcactgtgaaccttctccataaaccctaaactacctgcccccctccccttctggtctgtaggatgtgtccagtttccagcaggttgaaagacttgagagtggccgggggaaatgtccttttgagccagctcaacggtcagcagctgtaatggctggtgagtggggagagccaagagtcggtgattcttgctctaacgacctccttccccacccctgctactccaatttctgtgtctcttcttcccttgggatgtcaactgcactcatgtctccattcagcctttcctggtgtccccgcgtgactgtgacTACCAACATGTCTCCATAGGATGATATTTTCCCCCTGGCTCCTTTTTAGGGACTGTAGCCTCTGCA
This mutation is a 554 bp deletion beginning at Chromosome 6 position 82,907,725 bp and ending after 82,908,278 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65979 | GCC TGC TTC CTT CCA TCT C | Common | A | |||
65980 | GCA TCC CTA GAA TAG CCC TAG A | Wild type Reverse | A | |||
65981 | TGC AGA GGC TAC AGT CCC TAA | Mutant Reverse | A | |||
65982 | Fluorophore-1 | TCC AGA ATT TGA GAG GAA AGG TC | Quencher-1 | WT Probe | ||
65983 | Fluorophore-2 | AGG CAG TTA CAG ACT ACC AAC ATG TCT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65979 | 0.40 uM |
65980 | 0.40 uM |
65981 | 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 |