Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:99321872-99322023 152bp CGTGCGGTCCTAATTCAGAG CTCTTCCTCCTCCCATCCTC
Mut= 153 bp
Wt= 152 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AATCATGAAGTAGATTCAAGTATTTAACTTCATGTTAAACATGCCCTTGGCTGATAACTTAGTTGATTTTTATCATTAGCATTTTCTAACATTAAAAAAAAAACTTTACCAAAATAAATAAATAAATAAATACCACCTTTACcttttaaaattattgtgataaataatagcctttttaaaaagtcaaacccctggtttccagatcatcacctccaccaccagcaacgccaacgctgtactgcagattgacaatgccaggctcacggctgatgacttcagactcaagtaagtcagtgagcgaaaagaatccatggggattcgtgcggtcctaattcagagttgttcttaatccctccatcaaattctagtcagtcttactccaacgttagaaagcgcttggatttaaagtaagcaagatgaacctacaggggagaaaagactgttccagccagaggatgggaggaggaagaggaagtgctctagagaggccagccttagcCGAGGCATTCAAGGGGTACCTGAATAAAGTCAAGCCAATAAGATCCATCAAGATCCCAGCGGGCAGCACTGATTGGGCGCTAAGTTACAAAAAAACAAAAAAACAAAAAAAACAAAAA
This mutation is a 357 bp deletion beginning at Chromosome 11 position 99,321,844 bp and ending after 99,322,200 bp (GRCm38/mm10). In addition, there is a 10 bp deletion (ATCCCTTAAA) 160 bp after the 357 bp deletion, that will not alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39959 | CGT GCG GTC CTA ATT CAG AG | Wild type Forward | A | |||
39960 | CTC TTC CTC CTC CCA TCC TC | Wild type Reverse | A | |||
39961 | TGC CCT TGG CTG ATA ACT TAG | Mutant Forward | A | |||
39962 | CTT GAT GGA TCT TAT TGG CTT G | Mutant Reverse | A | |||
39963 | Fluorophore-1 | ATC CCT CCA TCA AAT TCT AGT CAG | Quencher-1 | WT Probe | ||
39964 | Fluorophore-2 | CCA CCT TTA CCG AGG CAT T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39959 | 0.40 uM |
39960 | 0.40 uM |
39961 | 0.40 uM |
39962 | 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 |