Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:105686820+105686979 160bp TTGCATGCTTCCTGTTTTCA TCCTTGGCTTTCCGTAGTTC
Mut= 126 bp
Wt= 160 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case and insertion with carrots ^ t ^ (t insertion):
TTAAATAAAAGTATTGTATTTTAATAGCTGTATTCCTTGTCAGACAGCTAGAACATATACCCCTAATTCTGCTAAAACTATGGAAAACGATCACCAGTTTGCTTGCAAATCAACTCTGCTGCATGCGTTCACAAGTTTC^ataaagg^TTGCATGCTTCCTGTTTTCAATACAAATTGTGTTTTTTTCATACCCATCTTTGCTTTTTATTTTTTTTATTTTTGTTTCCCTTttcttggttggatgcctgtgtgtgtttaatccaaatccctgtccagtcctggagtttgaactacggaaagccaaggagaccattcaggccctccgagccaacctaacaaaggctgcaggtggtgtacataaaactttttgagccttttcccacttttacaatgtagaagtagccggactatttttaagtgtaaaatagtaaaacattatctcattttatctcaccagaacatgaagttcctttacaggaacgaaaaaattataaatcaagtcctgaaattcaggtgggtgattgacaacaccagttatcttccatacaacccaagtgtcaaaggagcaaagctcacattttcccatctgtcttttaactttttttccaggagccaatcaaacctcttgaaaagagagctctcaacttcctagtcaatgagtttttgttaaagaataactacaagcttacatcaataaccttttcagatgaaaatgatgatcaggtacagttactgttttaagtaatgtctacttttaatctactgtttctctaacttgatttttaacacttatgcctgtaataagagtccatgtgggtagtttctaaaatcaaagcaggtactactcagtttagtattcaattggctcacaaaccttttctatttaaataaacctttttaaataaagacttatgtttctttccaggTAACTTTTTATAGCCTGATAGTAACTGTATAGCCCCTTCATGTTTGTATTCATTCATAATGTATGTTTTTCTAAGACTGAGTAATTACTTGCTTTTATTGATCATTGCAGACTTAGAAATTGAGTTTAACTATAAAAAAATTTCTTGAGTAGCACTGTATATAATTCAGATGTTCAGTAGCAATGGTTGGAGGTTTTTTT
This muation is a 696 bp deletion beginning at Chromosome 1 position 105,686,903 bp and ending after 105,687,598 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion and 7 bp deletion (ATAAAGG) 83 bp before the 696 bp deletion that will not affect the results of the mutation.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39187 | TTG CAT GCT TCC TGT TTT CA | Common | A | |||
39188 | TCC TTG GCT TTC CGT AGT TC | Wild type Reverse | A | |||
39189 | ACA TGA AGG GGC TAT ACA GTT ACT ATC | Mutant Reverse | A | |||
39190 | Fluorophore-1 | AAT CCA AAT CCC TGT CCA GTC | Quencher-1 | WT Probe | ||
39191 | Fluorophore-2 | TTG TTT CCC TTT AAC TTT TTA TAG CCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39187 | 0.40 uM |
39188 | 0.40 uM |
39189 | 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 |