Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:98554425-98554525 101bp ATGGGAGAAAATCTTGTGAAGG AAGTGACCAGGGTTAATGATCTG
Mut = 99 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTACATGAGTGTAGGGCACCACCTGCATACGTGCCTGGGAAGGCCAGGGAGTGAGTGTTGGATTCCCAGGAACTGGAGTTACAGATGGCTGTGAGCTTTCTGTGAGTTCTTGAAAGCAAACCTAGGTCTTCTGCAACAGCAGCAAGTGTTCTTAACTGCAGAGCCATTGCTCAGGGGCCTCTATCCAGACTTTTGATTGTTTTGTTTTTAAAGTTATTTACATCCTTTTGctggatgtaacttctgggttttttttccccttcttaggcttacctacaacaagttcaagatctggtgattttagagagtataattttgcagaccttaggtaagttaattttcctttttaaagcaggaaaaaaatatgtatgggatataatgatttcatgttaaagtttgttagatgggagaaaatcttgtgaaggctttgggaagtgtctgttcctgggtaaaaatgtctgTGGGGCTCATTAAATGGAGACAGATCATTAACCCTGGTCACTTTTCACAGTGTTAGGTCCTTGGTGACCCTTGAAACAGCTATTAGTCAAGATTTAAAAAAATTTTCTCATTGTAAGGTTTATGATTTTTTTAAAAAAAGTTTTATTATATATAAGTACACTGTAGCTGTCTTCAGACATCCCAGAAGAGGGTGTCAGATCTCATTACAGATGTTTGTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGTACCTCTGGAAGAGCAGTCGGTGCTCCTTAATTTTTATTTATTTATTTATTTT
This mutation is a 231 bp deletion beginning at Chromosome 15 position 98,554,468 bp and ending after 98,554,698 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48929 | AAG TGA CCA GGG TTA ATG ATC TG | Common | A | |||
48930 | ATG GGA GAA AAT CTT GTG AAG G | Wild type Forward | A | Wt F | ||
48931 | GGG CCT CTA TCC AGA CTT TTG | Mutant Reverse | A | Mut R | ||
48932 | Fluorophore-1 | AGT GTC TGT TCC TGG GTA AAA ATG | Quencher-1 | WT Probe | ||
48933 | Fluorophore-2 | ATC CTT TTG TGG GGC TCA TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48929 | 0.40 uM |
48930 | 0.40 uM |
48931 | 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 |