Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:31465595+31465754 160bp TGAGAAGAATCAAGCCTGCTG TGCCACGATAACCCATTACC
Mut= 160 bp
Wt= 160 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AACCTAATCACTGTAATCCTTCATAGGCACGTCTAGGCTAATCTAATCTAGAAGGTTCCTCACAGGTATGCCCTCCCTTGATTCTCCAGCCTCTTGAATGTAAACTCTGAGTAGTCTGGGAAGGCCTGGGCTGCAGAGGGATCAGTTGTGTAGCCTGCTTGCTCTGACTGCCCTGGGTgtaagaggaattgtgtacactgtgttgaattgacttgtaattgttagtatgttttaacccatgtacttttttttgtatgtggtttctttttgttttatttatttatttatttatttatttatttatttatttattttgctttttgtaaacaaaacaaagaccagtcttcatagttttacacaagttattagcttggttgtatttttacaatatgagaaaaattagtaggaaatgtctccagcttctcctgtgtggtccatgactttgtgttgtatcagtaatagggcccagtgctccataaaggataacagtttccagtacaccatccctcatgaggactccctgagtggctcgtcatccgcctcctcttgtgaaccagtcagtgacttcacagcaaccctccgaaaatctacctactggatgaagatgagaagaatcaagcctgctgccacttcacaggttgaaggtatccatttcagattctcagctctgagacaggagagtgtatctagtgtgcttgatattttggtatggtatggttgtttatcagtagtgaggtgagggctgggtaatgggttatcgtggcaatgtatgccagagagtcaccatcagtggttgatttatagctgtctttgttgagtgctgcttctgtagacccatttaagcataaggaagatctaatggtttcTGAGGGAAGAGGGGACTCCATGGATTAAAAACAATTCACACCAGTGGTGGCACACGCCTTTAATCCCAGCACTTGGGAGGCAGAGGCAGGTGGATTTCTGAGTTTGATGCCAGCCTGGTCTACAAAGTGAGTTCCA
This mutation is a 688 bp deletion beginning at Chromosome 5 position 31,465,168 bp and ending after 31,465,855 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42045 | TGA GAA GAA TCA AGC CTG CTG | Wild type Forward | A | |||
42046 | TGC CAC GAT AAC CCA TTA CC | Wild type Reverse | A | |||
42047 | CTG CAG AGG GAT CAG TTG TG | Mutant Forward | A | |||
42048 | TGG CAT CAA ACT CAG AAA TCC | Mutant Reverse | A | |||
42049 | Fluorophore-1 | ACT TCA CAG GTT GAA GGT ATC CA | Quencher-1 | WT Probe | ||
42050 | Fluorophore-2 | CCC TGG GTT GAG GGA AGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42045 | 0.40 uM |
42046 | 0.40 uM |
42047 | 0.40 uM |
42048 | 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 |