Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:162722804-162722959 156bp TGCCTACTAACTCCAAGAATAGAGTC AAGCAGAATGCTGGGTTCC
Mut= 150 bp
Wt= 156 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGTGTAACCCTAAAGGGCTTAGAACTTAACAGTGGATCAGGCCAGCCTCCAACATGAAGCTATTTTCCCACTTTCACCTTCCAAGTTGTGTAGTTTGAACTTTCTTCTCTTAACATTTTAAGTTTTTTACTTAAAATAGATTGCCTGAAAACATTTCAAAACTAGCATTTCCCCTTCCCATATTCccagtatatagtgttccatatttagaagttaaggggttctggtgggtgggtttgttttgttttttgagacaaattttgctggggtcccaagatggcttaatattttgagtggattgtttactttttaccttcaattaatgttgtaggttataaactgagtttatagagctctactttaacattggggactagtcagagattgttaactcctgttcatgtatattatttgttgaataaaatgtataatagagtgatgaacagtagatgttctaaggaaattgatgttactgatcttttagttgcagctcgacatgggttacaaagtcttggaaaagttggtatttcacggcgaatgcccccacctgctaaccttccaagtctcaaagcagagaacaaaggcaatgatcctaatgtgaatattgtgcccaaagatgggacaggatgggcatcgaaacaagagcaacatgaagaagaaaagtgagtcagaatctgttaagaataaaacacttctctcattgtaggaaactgccaccgctgtgcactgttcagccaatagaaacactctatgacacttataattcagagagttcttcctcagatgaaaacacaagtcagcttactctttgatgcctactaactccaagaatagagtcttggacttctcttaacactattacttagcaaaaacatgaaaaatgaacttaacattctaagaacaaatctacaagccaaccaatatttactgcaaactattatcTGAGTGTGGAACCCAGCATTCTGCTTTAATAAGTCCTTGATAAATGAAGAGTTAAAAAGATAATATAGATATTATAACTCTTTTGTGGTGTTATATTTAAAGTGTGAATAGACACATTTAGTGTAAATATATAATAACTGTGTATACACACTTTTAGTCGTTAGGCTCTTTGAGTTCAGTAGCATTAGCCATAAGCAC
This mutation is a 757 bp deletion beginning at Chromosome 1 position 162,722,830 bp and ending after 162,723,586 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39080 | TGC CTA CTA ACT CCA AGA ATA GAG TC | Wild type Forward | A | |||
39081 | AAG CAG AAT GCT GGG TTC C | Common | A | |||
39082 | ATT TTC CCA CTT TCA CCT TCC | Mutant Forward | A | |||
39083 | Fluorophore-1 | AGC CAA CCA ATA TTT ACT GCA AAC | Quencher-1 | WT Probe | ||
39084 | Fluorophore-2 | TTC CCC TTC CCA TAT TCT GAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39080 | 0.40 uM |
39081 | 0.40 uM |
39082 | 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 |