Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:63169613-63169703 91bp TCTGACTCAAAGCTTGTATGTAGGT CCAAAACGAACACATGAAACTG
Mut= 109 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case, insertions in^^):
CACCATTGTCATTTTCAGCAGGAGTCACTTCTCAATTCCCGGAGTAAAATATCAGTGTGGTTTGGATACTGCCGACTCATCTCGCTGTTACACTCAAACAATACCGAACCAAAGG^T^TTTACTTCGCATTCTGACTCAAAGCTTGTATGTAGGTC^ACCAAGGCA^tcctcaggaaacagctggagccatctctactcttgatattattcagtttcatgtgttcgttttggttgtagtttgttgtagttgttttgaggcagggcctcctatagctcaggctgacctccaatatgtaggctaggatgaccttgaacctctgacgctgccacctccacctcccctgtaaatcccaccatccctgacaactctccggtttctgctgtgtctgatggcgatatactcgtccttgcaggtggtattgtgaaggcaaggagcttgaaaactctccagatatccacattgtgcaggcaggaaaccttcactcactgaccatcgccgaggccttcgaagaggacacaggacgatactcatgctttgcttccaacatttatgggacagattcaacttccgctgagatttacatagaaggtaaagctaaatacttttggagtgtgacatgttctaactatctgtagttacgtaaacatttgtaggggggggcttttctacccatctagacactggggatactagtcatgaaccaagtagacaaaactcttcagcggatgtgccgatgaattcccgTTATAATGGGAGAAACTCCAAACAAATGaGATAAACCATGATAAATACATAACATTGCAAAAGAAATAGGAAAAGAGATGATCAATAAAGAGCTCACTCACCAGGTGACATTTGAGGGAAAAGCTGGTGGCCACAGGAGAG
This mutation is a 579 bp deletion beginning at Chromosome 10 position 63,169,099 bp and ending after 63,169,677 bp (GRCm38/mm10). In addition, there is a 9 bp insertion (TGCCTTGGT) at the deletion site and a single bp insertion (A) 38 bp after the exon deletion that will not alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39524 | TCT GAC TCA AAG CTT GTA TGT AGG T | Common | A | |||
39525 | CCA AAA CGA ACA CAT GAA ACT G | Wild type Reverse | A | |||
39526 | CTT TTC CTA TTT CTT TTG CAA TG | Mutant Reverse | A | |||
39527 | Fluorophore-1 | CAG GAA ACA GCT GGA GCC | Quencher-1 | WT Probe | ||
40474 | Fluorophore-2 | ACC AAG GCA TTA TAA TGG GAG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39524 | 0.40 uM |
39525 | 0.40 uM |
39526 | 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 |