Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:10047381+10047512 132bp TTGTGGAGTTCTCCCCTTTG GGCTGTTGTTGGCTGCTT
Mut= 96 bp
Wt= 132 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTACCTGGCTGATGAACTTCAAGATCTGTCTGTCTGTGATCTCTCAGAGTTTGGGTTACAGACACACGCTGCTGGGCCTGGCTTTTACATGGGTGTTGGTTTGGGAATACAAGCTCAGGTCCTCAGACTTGCACAGAGGGCACTTCACTGACTGAGCCGTCTCCTCATCCCCACTCTTTACATATCATGCATATGTAAATATTACATGCAAGTACAACATCACATAACTAAGAACTACATTTGTTTAGTGTctccagcctatcccctagcctcctggcccaatagcagaagcttcccaattttgtttttcactcttttctttctagtggctggcccagtgtacttaatagtaactcatttttaagaggatgaattaggtatcagaacagttgttacagtatgtaagtactttacacttcaatctctgtgctgtgtaattttttctaagttatttagtatgtcaaataagttaaattgtacattttgtggagttctcccctttgatttgtaaatatgtcttttccagggaaaggcgtcagagaaactctctgaaaacagtaaaatattaatctctatggctaaggagaacattccaccaagcagccaacaacagcccaagggacccttaggtatggtactgtatgcgtggaactaactttaagatatcctttctttgtagggcattgtgaaagaggtttgtttcttcagaaaaaagatttctttggatcttcatttttgagtcttacatatggcaatggggtttgtgtatgaaatgtaactcagagaacgttacctatttcattgatgttgcctaattgtgattggtggaaatgatattagaaaCTGTGTATGATGTAACTAAAAAATAGTAATAAAATTTTTAAGTGTGTTTATGTATATGTATACATGAGAGAGTCTGTATTGTTTTAAATTCAAAGAAGGGTCTAGCAAGATGGCTCAGCTGGTAAAGATGCTTGCCACCAAGCCTCAGGACCTGAGTTTGCCCCCCAGGACCCATATGGTGGTGTGCACACACATATACAAGTAAATGTGCTTA
This mutation is a 592 bp deletion beginning at Chromosome 1 positive strand position 10,047,149 bp and ending after 10,047,740 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38734 | TTG TGG AGT TCT CCC CTT TG | Wild type Forward | A | |||
38735 | GGC TGT TGT TGG CTG CTT | Wild type Reverse | A | |||
38736 | CCC CAC TCT TTA CAT ATC ATG C | Mutant Forward | A | |||
38737 | ACA TCA TAC ACA GAC ACT AAA CAA ATG | Mutant Reverse | A | |||
38738 | Fluorophore-1 | TGG CTA AGG AGA ACA TTC C | Quencher-1 | WT Probe | ||
38739 | Fluorophore-2 | ACA TGC AAG TAC AAC ATC ACA TAA CTA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38734 | 0.40 uM |
38735 | 0.40 uM |
38736 | 0.40 uM |
38737 | 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 |