Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:104940912+104941051 140bp CTGAAGGGGAAAGAACATTC CTGTCCATGTCTGAATGAAGC
Mut= 150 bp
Wt= 140 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACAATAATTTTAAAATATTAAGATCATACTGTGTTTGAATACCATAAGAATATGACATAGGAAAACTTCACATATTATTCTTTATCCATTGGTTCTTTACAGACAGACATCCTAAACACTGAAGGGGAAAGAACATTCTTATGTAACGGGAGATCTTAAGTCTGTTCCAAAGTGAAAATCAACTTATTTCCAatacctgtgcatatcaataatgacatttcagtgcttttgtccccagcttcattcagacatggacagaggagatggatccatcaaatacatcctttcaggagaaggagcaggcatcgtgtttaccattgatgacaccactggagacatccatgccattcagaggctcgaccgagaagaaagagcccaatatactctaagggctcaagccctagacaggagaacaggccggccaatggagccagaatcagagttcattatcaaaatccaagatatcaatgacaacgagcccaagttcctggatggaccttacatcgccactgtgccagaaatgtcaccagtaggtaagatggtggtgagctcatttttacaaatagtagatgcattgtaggagatgctactttgtacacatagaggagaaaaggtacagaaatcataaaagtgaaactaaaatgactaccaactccttcaataggtagcttcctgaagctatggagatatcagaagcagccacattaagtaataattttccaatgcctgttagttctgagttcttaagattcaaagacatgaacaatggctgatagattatattgtattagattagattagattagattagattagattagattgttaaggtaaatattggtattaccaataaacaaggaccacataggcagtgttacttttactggctactaagaatttaacacagctgggcgtggtggcgcatgcctttaatcccagcacttgggaggcagaggcaggtgaatttctgagtttgagggcagcctggtctacagagtgagttccaggacagccagggctacacagagaaaccctgccttgaaaaataaaataaaataaaataaaataaaataaaataaaataaaataaaataaaatttaaaaaaaagaaagaaagaatttaacacatatgactatcatccctattgagtaacaaaatgggagtcatggaatcgtgatcaagaaagggaactctcccagagaagcaagcaacatctgtaaAATGTTTTAAGGTTGACTATGCAAGTACTATACCAGCTGTTTTAAACTGGGCTTCATTAGGTTTCTCCCACTTCTCTCCCTCAGGTACATCTGTTATCCAAGTGACAGCCACAGATGCTGATGACCCAACCTATGGCAACAGTGCCCGGGTAGTGTACAGCATTCTCCAGGGCCAGCCATATTTTTCTGTGGACTCCAAAAC
This mutation is a 1028 bp deletion beginning at Chromosome 1 position 104,940,986 bp and ending after 104,942,013 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53808 | CTG AAG GGG AAA GAA CAT TC | Common | A | |||
53809 | CTG TCC ATG TCT GAA TGA AGC | Wild type Reverse | A | |||
53810 | GAG AAG TGG GAG AAA CCT AAT GA | Mutant Reverse | A | |||
53811 | Fluorophore-1 | ATT TCA GTG CTT TTG TCC CCA | Quencher-1 | WT Probe | ||
53812 | Fluorophore-2 | ACT ATA CCA GCT GTT TTA AAC TGG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53808 | 0.40 uM |
53809 | 0.40 uM |
53810 | 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 |