Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 116 bp
Wild Type = 98 bp
>chr4:134365004-134365101 98bp ACAGCAGGGGCAACCTCTT AAGGGAGCCTGAGACACCTG
Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):
ACAGCAGGGGCAACCTCTTGtgattgagggaatgtcactgtaaggtggcaggggccactggagctgcacggtgaggggcaggtgtctcaggctcccttcctgtaccttctcaggacctacccaggagagacccttcccaatgccaccttctgcctcatccctggtcaccgatctgccacctcctgctttctccaagcccttcaggtacaccccaatctggatattccccagcctcagtacctctgcccttgcactggcaggagattctgggcacatgcacataggaagggggttccctgtacccccaaatactcctctttgtcctttcccttaacaggggggctgatggtggcctggggtggtggtgaatgtgtgcaagcgctgggctccccaatgactgccctgccccccaggccggctgcatccccgtgctcctcagcccccgctgggagctgcctttctctgaagtcatcgactggaccaaggcagccatcattgctgatgagagactcccactgcaggtagctcagggtctgtgtcatggtggggggggggaatgtcaggggagggaccccaggatctcggcccccttctagtagtcattcttatacctattgcttccctctagggcagtcatagatgtagttgaagggaaggctctagagggtgctgagagcaagttctgaaccactttcttccttggttttcccatctgtataatgggataagttagatctctagggatcatctgaaagtaaacagataagatgccatgtctcttccatctGATGAAATGAATGTTGCCCAGGCCAGAAGCATCTCTGTGAGCAGCTGT
This mutation is a 767 bp deletion beginning at Chromosome 4 position 134,364,315 bp and ending after 134,365,081 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65440 | ACA GCA GGG GCA ACC TCT T | Wild type Forward | A | |||
65441 | AAG GGA GCC TGA GAC ACC TG | Wild type Reverse | A | |||
65442 | CCA GGT CAC TGC TCT TGG TTA | Mutant Forward | A | |||
65443 | ACA GCT GCT CAC AGA GAT GC | Mutant Reverse | A | |||
65446 | Fluorophore-1 | CCA CTG GAG CTG CAC GGT | Quencher-1 | WT Probe | ||
66294 | Fluorophore-2 | CCT CTT GGA TGA AAT GAA TGT TGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65440 | 0.40 uM |
65441 | 0.40 uM |
65442 | 0.40 uM |
65443 | 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 |