Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:101138886+101139008 123bp GAGCATATGAGCCATAGAATCTGA ACAGGAAACCACACCAGCA
Mut= 120 bp
Wt= 123 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATTCTAGAGACAGGAATTTTGCTGTATTACCTAATTCTAAACTTATGGGCAATTCTCCTGCCTCTTGCTCCTGAGTAGCTGGGACTGTATGCATACACCTTTGTGCCCAGCCATAAGTATTAAAATATGAGCATATGAGCCATAGAATCTGAAACAAAGTATATTCCTACATAACAGcttccaggggccttgtacttgttactgcaactgctgtgaaaaacggcagagactatgctggtgtggtttcctgtgttctgagctcataaagaggaatacacccccctccatatatcttactaggcatatatcttactaggcagtagctgtattgataaatttacatggggttttaaagttctgctcttgatgataaatttttgtttgtatttctaggactttcagtcagaagaaggctgctattgaaagagagtatgcacaggtgagtgtgggaattgcaatttctgtCTTAGGTATGCCTTTTCTTGGCTTAAGATCATGTCTCCTGTCAAAGAGAATCTTGGAATCTTCTTTGGCTGCCTGCCCATTTCTGACCATCTAGCCTTTGGTTTCCTTAAAAACAAATTTTTTTTTGGTTGTTTGGCACAAGGCTTCTCTATGGCTTGACTGCTCTGAAATTTGATCTG
This mutation is a 288 bp deletion beginning at Chromosome 7 position 101,138,935 bp and ending after 101,139,222 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42914 | GAG CAT ATG AGC CAT AGA ATC TGA | Common | A | |||
42915 | ACA GGA AAC CAC ACC AGC A | Wild type Reverse | A | |||
42916 | CAG CCA AAG AAG ATT CCA AGA | Mutant Reverse | A | |||
42917 | Fluorophore-1 | CTG TGA AAA ACG GCA GAG ACT A | Quencher-1 | WT Probe | ||
42918 | Fluorophore-2 | CCT ACA TAA CAG CTT AGG TAT GCC TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42914 | 0.40 uM |
42915 | 0.40 uM |
42916 | 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 |