Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:75291907+75292051 145bp TACAAGGAAGGGTTAAGGTTTG GAAAAACCACAGGAAGGTGTGT
Mut= 150 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGGTTTGGCTTAGCAAATTTTATTTTATAAAAGGAAATACAAGGAAGGGTTAAGGTTTGTAAAGTTCTCATTATATAATATTTTTGTATGTTCAATGTATACGATGTATTTCGAGCATATTAATACCCCattccaggaactactctctacctaaacacacacacaccttcctgtggtttttcattcccttatggtgttattattattggagtggcatatgcttagctggcatttgggtctctctgacttgattttgccctggtaccatcttttctccagacatttacatagtaatatttgcctcatagacacagtgtcgtaactggctgaagactggcagctgcctctatggaaacacatgtaggttcatacatggcccttcacctcgtgggaaaggctacagcagcaattacagaaggtaaactgagaactcagatattttctatagtattaaaaaccaacttttgtagtttagtgtaaaaatatgaaataaaaccatatatttggtcagattttctttttttggatagtcttattttggttgttactgttaagattaataaaactttagctttaaagtgtacatgtttacatgttttggtagtagatccatttttcaatgtgtatattcaaatcaatgaacacctgtggagagactgagtctgggaaagaacagcccttaaagagcccctACCTGACACAGCCCCAGGTGTGTATTAAATTTCTGCTTAGGCACAACTCAGACGTTTGAGAGAGTTTACTTTTCGTAAACGAGTAAAATTTAAATAAGGTTTAATTGTAATTTTCTACAGGCATTTTAAATTCATATCT
This mutation is a 563 bp deletion beginning at Chromosome 14 position 75,291,999 bp and ending after 75,292,561 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41660 | TAC AAG GAA GGG TTA AGG TTT G | Common | A | |||
41661 | GAA AAA CCA CAG GAA GGT GTG T | Wild type Reverse | A | |||
41662 | CAA ACG TCT GAG TTG TGC CTA | Mutant Reverse | A | |||
41663 | Fluorophore-1 | CCA GGA ACT ACT CTC TAC CTA AAC ACA | Quencher-1 | WT Probe | ||
41664 | Fluorophore-2 | AAT ACC CCA CCT GAC ACA GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41660 | 0.40 uM |
41661 | 0.40 uM |
41662 | 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 |