Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 139 bp
Wild Type = 141 bp
>chr12:55841784+55841924 141bp GCCAGGAGGCTTCAGTTATTG GTGCAGCGATGGAAACACT
WT sequence (deletion in lower case, ^G^ is an insertion):
tggatgtccagtttcatattacaactttttttttttctgtctggactaacatcagaattttttgaaacagttttatcttttctgtttgtttttattttttgagatagggtttcatgtatcccaggctggcctcaaacatgctaagtggcgaatagtcatagctagtcttagactcctaatcctgcctgtgcctcccaagcgctggaatctcaagtgtgtgcctccaggcctgccctgaagttttatctttgatatttccttttttttttttaacagaaatggatgatgaagattgtgaaaggagaaggatggagtgtttagatgagatgtccaacctggagaagcagtttacagacctcaaagatcagtaagtactaaccagatggccctcgtcactgaatctcagtggcatcagttttaccagcacaggagcaattacagcgtttctgctgtgtttccttttgtgttttatgggaactggctgatctggccaggaggcttcagttattgtttacccgctagagacctcattagctcaacagagttttgaggccagtgaggtTTTGGGGGTGGATGGAGTAGGTGACTTGCATGGGCACGCTGCTGATTCGAGTGTTTCCATCGCTGCACACTGCTTCTGTGTGTGCTGTTCATTCCCTTGTTA
562 bp deletion beginning at Chromosome 12 positive strand position 55,841,295 bp and ending after 55,841,856 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000654815 (exon 2) and 471 bp of flanking intronic sequence including the splice acceptor and donor. In addition there is a single bp (G) insertion at the deletion site
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37223 | GAC ATT ACG TTA ATT CTA TGG TGG T | Mutant Forward | A | |||
37224 | GTG CAG CGA TGG AAA CAC T | Common | A | |||
37225 | GCC AGG AGG CTT CAG TTA TTG | Wild type Forward | A | |||
37226 | Fluorophore-1 | CTG GAC CTG TTT GGG GGT | Quencher-1 | MUT Probe | ||
37227 | Fluorophore-2 | CCG CTA GAG ACC TCA TTA GCT C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37223 | 0.40 uM |
37224 | 0.40 uM |
37225 | 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 |