Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 88 bp
Wild Type = 148 bp.
>chr13:94978617+94978764 148bp CTGACAAAGACGATTTAGAGCA ACCATCAGGGTTCAAAGTCC
Wt Sequence (deleted region in lower case, insertion in carrots)
TCATGAGGGTCCCCGGGACGGAGCTCAGGTCATCAGACTTAGTGACAAGTGCCTTTACCTGCTGAGCTAGCTCCTTGGTCCTAACAAACTTTGATTGAGAAACAACAGTTGAGTTGA^GTTGA^TTGGCCCAgtagttttaaaGTCGGGTTTATTGAGACTCAACAAAATAAAGTTTACTCTTTACAAGTTTATTGTGGTTTGAAAACACCAAAGGTTTTGATAAGAGAAATACTTCCATgtagaactggtatgtattagcattagaacataatatcttgtggcttctaaaagcttttgttcttgcatggatactgtgcagttaaaatcattttatgcttatgttttgtgttttgtgggtttttttttttttcattacaagaaagctgctttgcagacaataggagaggaccaaggtcagaacccctatactgaactgctggtactggaggctcatcgcgacattgtccgcttcctggtgaggctcgatgacttcaggtaagaggcttactctgcccagtggtcaatgctgacaaagacgatttagagcattttatgtttaaaacaacttcaaacttaaattatcttttgtaaaaactttCAGAATCCTGAAATATAACCAACTGAAAATTGTAGCATTTAATATTCAGTTGGAATGGACTTTGAACCCTGATGGTAACATGTTTAAATCCTTTGAAATCCATGTCACCTTGTCAGAGTCTGGTTGTCTTGACAATTACATTTGTTTGCTTTTTTTTTTCTTTGATGTTTTGGAAAAAATTGTCATGAGGTTTTAGAAATATTCTTACTACCTTAGAAGAAGCAAATGACTTTAAGATTGAGTTGTAGCTGTCAAATCTGAGAATCAAATATGTATGGAGTTTAATTTAATATTAGATACCCGCTA
391 bp deletion beginning at Chromosome 13 positive strand position 94,978,329 bp and ending after 94,978,719 bp (GRCm38/mm10). In addition, 96 bp before the 391 bp deletion there are 2 small intronic indels, a 5 bp (GTTGA) insertion followed 8 bp after that insertion by an 11 bp deletion (GTAGTTTTAAA) that will not alter the results of the exon deletion
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37958 | GGT TTG AAA ACA CCA AAG GTT | Mutant Forward | A | |||
37959 | ACC ATC AGG GTT CAA AGT CC | Common | A | |||
37960 | CTG ACA AAG ACG ATT TAG AGC A | Wild type Forward | A | |||
37961 | Fluorophore-1 | AGA GAA ATA CTT CCA TGT AGC ATT TAA T | Quencher-1 | MUT Probe | ||
37962 | Fluorophore-2 | CAG AAT CCT GAA ATA TAA CCA ACT GA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37958 | 0.40 uM |
37959 | 0.40 uM |
37960 | 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 |