Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:3223168+3223326 159bp GCATGCTGCTGTGAGAGACT GATGGCTGTTTTACCCAAGG
Mut = 159 bp
Wt = 159 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case;):
TATTTAGAATGCTGGGCATGCTGCTGTGAGAGACTAGCTTCTCCTCTAAggggaggggtgggaatccccttgatataagagcatttcatacctgagcatcaggggctaccagccactgaagggctagcgactgtcatggatgggagtgacggtaccttgggtaaaacagccatcgtgtctctgaccagccactctgtctcgcaggcaggaagagggttttgttttttgttttttaagtcattacattctcaactcattattcctattcttacatgaaggaattcaaagctctaaaatgcgattttctgaccttctaggtcccgttatgacacaggacattaccacgtatcacacggtgtttcttttggccattttaggaggaatggctttcattcttttggttttgctgtgtctccttttatattattgcaggtaaagtgttaccatttggatgatgtatttgtttaatgtagaatttcctatgcaaTTGGTATTCGGAAATGTTTTTTTCCTTCTTCTTTCTTTTTTCCCC
This mutation is a 438 bp deletion beginning at Chromosome 2 position 3,223,202 bp and ending after 3,223,639 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50094 | GCA TGC TGC TGT GAG AGA CT | Common | ||||
50095 | GAT GGC TGT TTT ACC CAA GG | Wild type Reverse | ||||
50096 | TAA CCT GTG GGG TCA TTT GC | Mutant Reverse | ||||
50097 | Fluorophore-1 | TGA GCA TCA GGG GCT ACC AG | Quencher-1 | WT Probe | ||
50098 | Fluorophore-2 | TCT CCT CTA ATT GGT ATT CGG AAA TGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
Primer 1 | 0.40 uM |
Primer 2 | 0.40 uM |
Primer 3 | 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 |