Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:56912825+56912982 158bp GTGATTTCCTGGATCTAAAAATGG AACAATGGTTCCCTTCATTG
Mut= 168 bp
Wt= 158 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (GCATGATT insertion):
TTATAGATATGAGCCACCATGCTTGGCAAGTGGTGCATTTAAGTAGACTATTTTGTGTAGTAATCTCGTTTACCTATTAATCAGTTGGGAAGGGGAATTGAACAGGCAGTGTCAGTTTAAATAAATAATTATTCTTTTATTCCATGTTGTTATAGAAATTTTATTTTAATGTTTCCAATATCTGAAAGCCATGTTAATGTATTTGAAGATAAATTAATTTTGACTTTTAATCTTTTCTGAGAGTTCATATTTTATACTAAAAGTGATTTCCTGGATCTAAAAATGGTGTTTGATTTCCATGAT^ctaatctgtggttaaaatatcagttataacctttatttttggtttcttatgaaatggagctttaagttgtacctcttgtttttcagagatataactacaatgaagggaaccattgttgctcaagtggactcaagtgagtccttccaagaattttgtagtacatcttgtttgtctctctatgaggacaaacagagtcctgctaaaggagctctaaataaatcaagatgcacaatctgtggcaaattaactgaggtttgtacttctgtacttttcatttacttgtatgagaaaagagttcagattttaacagttttcctgtac^TCTATATTGATGCATGTATTCTCAGAATGATGATATGCAAGACTAGGATTCATAAGACTAGAATTGTATAATTCACCCAAAAGAGTAAGTAGTATTATGATTGCTCTTTTTGGTTGTTGTTTTTTGTTTTTTGTTTTTCGAGACAGGGTTTCTCTGTATAGCCCTGGCTGTTCTGGAACTCACTTTGTAGACCAGGCTGGCCTCAAACTCAGAAATCTGCCTGCCTCTGCCTAGGATCAAAGGCGTGCGCCACCACACCCGGCTTGCTTTTTCTTTTTTATTGGTCTAATAATATGTTTATTTATACAAATAGACTGACTATACAAGAAA
This mutation is a 321 bp deletion beginning at Chromosome 14 position 56,912,866 bp and ending after 56,913,186 bp (GRCm38/mm10). In addition, there is an 8 bp insertion (GCATGATT) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48699 | GTG ATT TCC TGG ATC TAA AAA TGG | Common | A | |||
48700 | AAC AAT GGT TCC CTT CAT TG | Wild type Reverse | A | |||
48701 | CAA CAA CCA AAA AGA GCA ATC | Mutant Reverse | A | |||
48702 | Fluorophore-1 | ATG AAA TGG AGC TTT AAG TTG TAC CTC | Quencher-1 | WT Probe | ||
48703 | Fluorophore-2 | ATG CAT GAT TTC TAT ATT GAT GCA TGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48699 | 0.40 uM |
48700 | 0.40 uM |
48701 | 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 |