Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:76652261+76652355 95bp GTCATGGAGAGCATCCGAAC AAGCCCTGAGTGTATCTTACCC
Mut = 96 bp
Wt = 95 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TAGAAAACTAAACAAGTCGTGCCTCCACTCTATAGAGCAAGAAAAACAACTGAAGAGAGAGGCAAATTAGTCAAGTGATTTTGTTTCTATTTTCTGATTTTCATTATTCCTACctcagattcacacacgtcattaagatgggccttaaataacacaaaaaggtaccttcagtcacatgatcacatattagagcatcttagacacatgtctttttttattctagaaaaattaactttgaccacagtttgatccgccaatcaaggagcagcaggaaggcggtgctgtgcgtggtcatggagagcatccgaaccacacgccagtgctctctgtcagtgcacgctgggattcgtggggaagccatgcgggtaagatacactcagggcttcactaacgacacagttactatggagtttaaaaaaaaattactcatcaaaggcagaaaaaaaaaaaaaaaaaaagagcctcagttttgtagggcttcagtccggagtggctgtgctttggaataagaaaaccaaccaaaaccacttgggttctaaccctgtgtgctacggtacaaacaatatattactggagtctctccaattaactactacctcagtgttagagtaacCACATAAAGTAAGCCTTGATGTCGTATGCTTTGACATTTATTTATTTATTTATTTATTTATTGTTTTTTGAGAAAGGGTTTCTCTGTGTAGCCCTAGGCTATCCTGG
This mutation is a 500 bp deletion beginning at Chromosome 2 position 76,652,084 bp and ending after 76,652,583 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50905 | GTC ATG GAG AGC ATC CGA AC | Wild type Forward | A | |||
50906 | AAG CCC TGA GTG TAT CTT ACC C | Wild type Reverse | A | |||
50907 | CTG AAG AGA GAG GCA AAT TAG TCA | Mutant Forward | A | |||
50908 | AAA GCA TAC GAC ATC AAG GCT TAC T | Mutant Reverse | A | |||
50909 | Fluorophore-1 | CTC TGT CAG TGC ACG CTG G | Quencher-1 | WT Probe | ||
50910 | Fluorophore-2 | TTT CTG ATT TTC ATT ATT CCT ACC ACA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50905 | 0.40 uM |
50906 | 0.40 uM |
50907 | 0.40 uM |
50908 | 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 |