Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:31674438+31674540 103bp TCCCTTCTGTTTGTTCAGCC CAATGTCTCCGACCTCTCCA
Mut= 103 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CACCATTAGAACTCAGTGAGCTCACTGTAATTGAGGGAGCTCTGACCAGAAAAGAGACACAGTCCAATGCCTGGGCCTTAACAAAGGGCATTTCACCCTTGGAATCAGGGTGAGGGACCAGCAAAGTGTCTGAGCCAGGCAGGCAGTGTGGACATGTAACAGCTTGCAGTGAGCAGATGCTTTAAGGATAGCTAATGAGAGTCTGAAGTTCCCAGGTCTAAACAGTAATTATACCCATGTGCTAAAAAAGATGGTTATTGCCTCCCTTCTGTTTGTTCAGCCTCAGACACTGTGTGGCCAGCCCGCTggctattggtaagcttctgttttctcttgcagatacaatggagaggtcggagacattgtagtggggagaatcacagaggtaaagtcagtgttcataaagagaaggaggctggcggctcagggattggggccttcatctgtgaaagtatgaagaaggggccctttgtttctggggtggtgtgaaatgctatcccacatttatttctctcacaggttcaacagaagaggtggaaggtagagaccaattccaggctggactccgtcttgctcctctcatccatgaacctacctgggggagagctggtgagtgctcgcctgctctggaactgtccggtcggtcgggctgtggggctttgctccacctgagaagtctgcttcaCATGAGGTTGCTGTGGTTTTGTCACCAGATCTGCTACTTTGGTGAACACAGAGGCTTTTCTTCTAGCCCTGTGCTCTCAACATGGGCAGCAGCTTGTGTGTGGTAAGACCAGAAACACCCATGGCGTGGGCTCCCAGGAGGGAGCTGAGAGGAGGCTCTTGGGTCTTAGGATCTTAGTGCTGATTTCCTTTCTTGATGCCCAGCCTTGCACAGGGAAGGTGTCTCTTGAGGGGTTTGCCTTAGCTTGCTTGCTTTTTGGAGTGTGGGGTGGGTGGGGTTGAGACAAGGTTTCTCAGTGTAGCCCTGACCTGGAACT
This mutation is a 378 bp deletion beginning at Chromosome 2 position 31,674,483 bp and ending after 31,674,860 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48640 | TCC CTT CTG TTT GTT CAG CC | Common | A | |||
48641 | CAA TGT CTC CGA CCT CTC CA | Wild type Reverse | A | |||
48642 | AAA AGC CTC TGT GTT CAC CA | Mutant Reverse | A | |||
48643 | Fluorophore-1 | AAG CTT CTG TTT TCT CTT GCA GAT AC | Quencher-1 | WT Probe | ||
48644 | Fluorophore-2 | CTC ATG AGG TTG CTG TGG TTT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48640 | 0.40 uM |
48641 | 0.40 uM |
48642 | 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 |