Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:105805212+105805336 125bp TCTGCTGGTTTCTTGCATTG GCTCTGATACTTCTCACATCCAGT
Mutant= 110 bp
Wild Type = 125 bp
Wt Sequence (deletions in lower case and insertions with carrots ^ctggttttgggagt^ (ctggttttgggagt insertion):
CTAACATTTTTATAATTCCAGTTCTTAGATAACAAAAAAAAAAAAACCAGAAAGACCCTCTGCATAAATTAACAAATAACTGAGAACTTTGTTGTTCTGTCATTTGAATTGGTTTCCCTGC^ctggttttgggagt^tttactaacaaccattgtttgttaacagaatctcccacaatctgtaattgaaaatgttggagggaagatttttacatttggatcttacagactaggagtccacacgaaaggtaggtaatactttgcttattttgagctagtagaatttgcatttctttaagattttgagttattaatgactcataaacgagctttggttattgtggacgtgaatccttaagaagctgtctgtagctgccatctgtaatgcagtatagtaagtaatacgctcagttaaccagtggagtaaggtgtgttttctgtatctaagcactctgctggtttcttgcattggaaagtgtgggcacagagcaaattccactgtggtctatggACAATTCTCCATAGATTCCTTCTTTCTTTCTGAAGAGTATCACTGGATGTGAGAAGTATCAGAGCTCTAAGAAAGTATCGTCATTTTCTTGTCCTCATTTTTTTTATTGTAGATAGCTTATAAAATACATCTTAGCAAATACAGAATCAGTATGCCTGTAGCTACTGATCACATTGAATATTTACTTTAAATTTTT
This is a 373 bp deletion beginning at Chromosome 12 position 105,804,899 bp and ending after 105,805,271 bp (GRCm38/mm10). There is a 15 bp insertion (ACTCCCAAAACCAG) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47860 | GTT GTT CTG TCA TTT GAA TTG G | Mutant Forward | A | |||
47861 | GCT CTG ATA CTT CTC ACA TCC AGT | Common | A | |||
47862 | TCT GCT GGT TTC TTG CAT TG | Wild type Forward | A | |||
47864 | Fluorophore-1 | TGG GCA CAG AGC AAA TTC | Quencher-1 | WT Probe | ||
51332 | Fluorophore-2 | CTG CCT GGT TTT GGG AGT AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47860 | 0.40 uM |
47861 | 0.40 uM |
47862 | 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 |