Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:155961315-155961401 87bp TCTCTGTTGTGGGGGATGTT ACAATGCTAGGTCAGGCTTCT
Mut= 87 bp
Wt= 87 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
CTTTATAGCAAGACCCTACCTTATTTTACCTTATTTATTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTGTCTCTCTGTTGTGGGGGATGTTCAGGCCATCGgtgtgaactgagtatccttcttttataaatggcttgagaagcctgacctagcattgtaagtgcattggataagggctgaagtggttctcaagcctttgatcatgtttgggagcacatgaaactctatagtttctgaagttaacttcagatctctgactctattactatcactttttctcagtagatgtaaccacatcatgggatgcactttctcagaccaaggctgctgtaagtagttttagtttctattttaaacatttacttggtttaaaattgatactatgttatgatgtgattaattagaatatttaattggaaaacaaaggcatagattacacagtgaacaaatgtgagtagtgaagaagtaaactagacactgcagtagggtctcgGGTCTGCTACTGTCATGACATGTCAAGTATTGGAGTGACCTGTGTGGCTCCTCACCTTTAATTAGCTGGTTCACTTTGTGAGTTGATAggtaGGGTGGATATGGGGAGGTTGGATGGTACCTGTGTAGAAGGCTCATTATCAGGG
This mutation is a 392 bp deletion beginning at Chromosome 1 position 155,960,980 bp and ending after 155,961,371 bp (GRCm38/mm10). In addition there is a small indel 1 bp (T) insertion and 4 bp (GGTA) deletion 88 bp after the 392 bp deletion that will not alter the results of the deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39560 | TCT CTG TTG TGG GGG ATG TT | Common | A | |||
39561 | ACA ATG CTA GGT CAG GCT TCT | Wild type Reverse | A | |||
39562 | AGG TGA GGA GCC ACA CAG GT | Mutant Reverse | A | |||
39563 | Fluorophore-1 | CTG AGT ATC CTT CTT TTA TAA ATG GCT T | Quencher-1 | WT Probe | ||
39564 | Fluorophore-2 | CCA TCG GGT CTG CTA CTG TC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39560 | 0.40 uM |
39561 | 0.40 uM |
39562 | 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 |