Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:43604489-43604588 100bp GGATGTGAGCAGGCTGTGTA ATGGTTTGCCTCCTGTGCT
Mutant= 100 bp
Wild Type = 100 bp
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):
CAGGAGGCAGAGGCAGGAGGGAGGCTTGAGTCTGCCAAAATGCAGTGTTTGTTGCTAAGTCTCTCTGGTCTTCTATACCATATGAATTTTAAGCCAAGTATAGATTACTCTATACAATTAGAGGAAAGTACACTTTT^c^acatggtagaattcaaatgcaattaaagaatgtcatttctcttaaatgaattctagggctctcggatcggacactggtgggtccaccaggaaccctgctgcccactgtgggattgttggtttcaaaccaagctatggcttggtttcacggcacggccttattcccctggtgaattcaatggacgtgccaggaatcctcaccagatgtgtggacgacacagcaattgtgttaggtatttctgcagttgcaaagcctatttgaaagcctcactgtaaaacattctgtctgtcacagtaagggtttctccttgcaggtacactggctggacatgaccccaaagattccaccacagtgagaaaccctgctcagccggcctcggttcccggcgggatggatgtgagcaggctgtgtataggaatcccaaaggtaactcgcttctgttgctttacaaaaagctgggaagttagatgcagagcacaggaggcaaaccatgaccagcctggtgggtttgaatctcttagccactacctctacaggccttgtgactgctgtcggccaagccagcccagtgtctccccttgagaaatggggatagaatccagatcctgagagttgagaggattaaatttgattatcctataaagcaaggctctgtagactttccttggaaagaaagcaacaccacgtTAGGGTTTGTAGATCGTAGATCTGGCCATTATAGTGAAAAAAGCAGCCATAGGCAGTTTGTTTATTTACATAAAGAAATATGTCTGTATACCTATAAAATTTTCTTTTCAAAAAGTGGATACACGGTAATATGTTGCCTGTGATCAGTGTGCTGATCCTTACTGTAAAGGACATAGGCATTCCTTCTTTAGTAAGGCCCC
This is a 687 bp deletion beginning at Chromosome 10 position 43,884,488 bp and ending after 43,885,174 bp (GRCm38/mm10). There is a single bp (C) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49029 | CTA AGT CTC TCT GGT CTT CTA TAC CAT | Mutant Forward | A | |||
49030 | CGA TCT ACA AAC CCT AGA AAA GTG | Mutant Reverse | A | |||
49031 | Fluorophore-1 | TCC CAA AGG TAA CTC GCT TCT | Quencher-1 | WT Probe | ||
49032 | GGA TGT GAG CAG GCT GTG TA | Wild type Forward | A | |||
49033 | ATG GTT TGC CTC CTG TGC T | Wild type Reverse | A | |||
49034 | Fluorophore-2 | AGC CAA GTA TAG ATT ACT CTA TAC AAT TAG AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49029 | 0.40 uM |
49030 | 0.40 uM |
49032 | 0.40 uM |
49033 | 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 |