Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:20114348+20114479 132bp AAATCCTTGTAGATATTTTCTGTGG AAGCCCTAACGCCATGACA
Mut= 105 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTCCAGGGTATCCAGTATCCAGTCCCATTCTTTGACCTCCGTAGAAACCAAGCTCATGGGTGGTACACATACATACATGTAGGCAAAACACTCATACATGTAAACCTAAAACACTTTAAAAGAAAAATCCTTGTAGATATTTTCTGTGGCATAATGATGCCACTAATGAATCATTGCAAAGTAAATCACTGGCAGcctgtactgttcagcatatctgcaaggacaaatttaattgcctgtcatggcgttagggcttgattatattttaacagtcttgattatattttaatataccacttaactattatttgtgcattcttaacaataactattttaggtctactggctattcagaggtgatagttgtggttggaggctgtgaacgagttggaggatttaatcttccatacactgagtgctatgatccagtaacaggagaatggaagtccttggccaagcttccagaatttaccaaatccgagtatgcagtctgtgcactgaggaatgatattcttgtttcaggtaaatacaaattattatagtagctacaacttttgattgggaatcagattggctgtttttaaatgataatgacttactgagtgtaaaatgaatatttcagataaacaagtttatacaacaaaactgaaaagactttttgcccttgaaagcAAGAATACTTACAAAAGTTCTACCAAAAATTGGTTTGGTCATAATATTTTAAATGCTTTTATGTACATTTAGAGTATATGTATTTTAACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTATATATGTGTGTGCCCAGAAGAACATAGAAGTG
This mutation is a 476 bp deletion beginning at Chromosome 16 position 20,114,420 bp and ending after 20,114,895 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44142 | AAA TCC TTG TAG ATA TTT TCT GTG G | Common | A | |||
44143 | AAG CCC TAA CGC CAT GAC A | Wild type Reverse | A | |||
44144 | CCA ATT TTT GGT AGA ACT TTT GTA AG | Mutant Reverse | A | |||
44145 | Fluorophore-1 | CTG TTC AGC ATA TCT GCA AGG A | Quencher-1 | WT Probe | ||
44146 | Fluorophore-2 | AAA GTA AAT CAC TGG CAG CAA GAA TA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44142 | 0.40 uM |
44143 | 0.40 uM |
44144 | 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 |