Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 93 bp
Wild Type = 93 bp
>chr19:38865790+38865882 93bp CATTTGCCAGGTGAGACCAT CCCATTTTGTAGCCCATGTT |
Wt Sequence (deletions in lower case):
CCTGAGGGTCAGATATTCAGCTTGAAAAAAACAAATCTGTATGGACGATTCTACCaaaacatttgcataagtcagatagtctgaactgacttattccaagcagtgttggtataagtattagggataatttttatctaaaatattttaaatttgctttggaaataaatctgcttcttaacttattaattaatagttccctagttatggaaaacaattcatgtgaattttctgtgtttcagaaaccttttcccaaaaaggacaaaggaactcaaatcagttgtccatagtgctcctggctggaagttatttggtaaagtccctcccagagaaaatcttcagaaaacctccaaaattattcagcaggtaagtactaaaatgactgtgaagtttcccagggctcttacagcattgctcatttgccaggtgagaccatgagagtctcaggtggcagggtcaccccaaatttgtACATAGTGAAAACAAGCCAACATGGGCTACAAAATGGG
This mutation is a 413 bp deletion beginning at Chromosome 19 position 38,865,432 bp and ending after 38,865,844 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64766 | CAT TTG CCA GGT GAG ACC AT | Wild type Forward | A | |||
64767 | CCC ATT TTG TAG CCC ATG TT | Common | A | |||
64768 | CCT GAG GGT CAG ATA TTC AGC | Mutant Forward | A | |||
64769 | Fluorophore-1 | CTC AGG TGG CAG GGT CAC | Quencher-1 | WT Probe | ||
64770 | Fluorophore-2 | TGG ACG ATT CTA CCA CAT AGT GAA AAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64766 | 0.40 uM |
64767 | 0.40 uM |
64768 | 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 |