Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut= 98 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
>chr9:27317023+27317120 98bp CACTGGGAAGGAGCTAAGTTCA CTGCTGGAAGGGACAATCTG
Wt Sequence: (Deleted region is in lower case)
ATCCCTGGATATGGCAGTCTCTAGATGGTCCATCCTTTTGTCTCAGCTCTAAACTTTGTCTCTGTAACTCCTTCTATGGGTGATTGAATGAAAGCATTTCTAAAAGTTCTTTCAAGCTGATTATCCAAACAGGATAGAATGTTAATCACACCTTCAGTGCTTGGGGGAGGATAACCGCTCATGTAATCAGGGGAGAATTAAGAAGGTAGAAGCCTGTAGAATTCACTACTCACCCCTTTATCCATAGGAGCCAAAGAGGAGGCAGGCAGGTGGGAAATTGTTTTCCGTTGTCTGGAGAATGTGGTAAAAGTAAGACTGTGCATCATCCTTAGCCTGGGACCTGTATTAGGCCTTTATGAGACTGTCAGGAAATCTGCAAATGAATGGGTTTGGTGGAACTGGGATACAGGATGTGGGAGGCTCTGTGCAGTGTGATAATCCTGAAGGGAGGTGGGAAATTCTTCAGCCTTGTTTAGGTCTTGTTCCTAGCGCTGAGCGTGGCAGCTTCCCAATAGCGGGATGGTTCACTGGGAAGGAGCTAAGTTCAGCCAGATGAGTATAGAATTCGGAGCTAGccagactgaggaaaggtccaaagttgaccagattgtcccttccagcagctcaggcacagggtgctgattcttttaaaagttggtaatccaaacaggatgggctataattcgaactgcagtgcctgggagaggctaatcagtaggttctaggtctctccatctctgtgatatactgggtaggagtttctgcactctttgttcttgacctcttgtccttgcacgcagcagagttctgcacgagtagatggagtccgcctctcattttcctgttgtcctttgcagcccctcccacctttacagaaacacccccccagtacatcgaggccaaggaaggtggaagtattaccatgacgtgtactgctttcgggaaccctaagcccatcgtcacctggctcaaggaagggaccctcctcggtgctagtgcaaagtatcaggtgagtcagctctcacctgagtgccacagccacgcttcctgaccctctctccctgcccatctcccatggaatgcagctggccaccgaaggtatgcgtacacggcccagggtggaggcaggcaacctcatggcctgacttgactacagacattctgctgcctcttgtagtgaggcccttgtcctccctgcccctgtacgtgagTCTTTGAGGCTATTTCCAGCTTCTCAGAGCTCATCTTCTCACTTAAGAGGCATCCTGGTGAGGCTCACAGGTTGATCTTGAGTTCTTTCTGTGTCCCTAGGTGAGTGACGGTAGCCTAACGGTGACGTCAGTCAGTCGGGAGGACAGAGGCGCCTATACGTGTCGAGCATATAGCATCCAGGGTGAGGCTGTGCACACAACCCATCTGCTTGTTCAAGGTAGGGGGACTTTTTTAGCCTCTTTATCCCTGGAACTCTGCAGGAGGCTTCCAGGCCTTTTGAGTCATGGAGAATGTTTTTCTCATGGCACAGTCTGCCCACTTGGGGGTGGTAGAGGAGCTGAGTGGGACTGTCTTCACGTGTGTCACTTACCATGTCTCTCTCACCTTGGTCAGCTGGGAAAAGAGCATCTCATGGTGTGGCAGGGAGACAGGCATGCTGAGCCATTTTTGATATTCCGTTGAGAGAGCATTTCTACCATGTCTCCACCTTCCTCAGAGAGGAAACACCCTTGGGTCATCTGTCATTCAGTGATGACATCTATT
This mutation is a 636 bp deletion beginning at Chromosome 9 positive strand position 27,317,073 bp and ending after 27,317,708 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37758 | CAC TGG GAA GGA GCT AAG TTC A | Common | A | |||
37759 | CTG CTG GAA GGG ACA ATC TG | Wild type Reverse | A | |||
37760 | TCT TAA GTG AGA AGA TGA GCT CTG A | Mutant Reverse | A | |||
37761 | Fluorophore-1 | AGA CTG AGG AAA GGT CCA AAG TT | Quencher-1 | WT Probe | ||
37762 | Fluorophore-2 | CTT TGA GGC TAT TTC CAG CTT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37758 | 0.40 uM |
37759 | 0.40 uM |
37760 | 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 |