Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:191582471+191582610 140bp CGTTCCCTATCCTCATCAACTC TGGACCAGAAGAAAAGACACAG
Mut= 140 bp
Wt= 140 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CATCAGATCTCATTACAGATGGTTGTGAGCCATGTGGTTGTTGGGACTTGGAACTTAGGACCTTTGGAAGAGCAGTCAGTGCTCTTAACCACTAAGCCATCTCTCCAGTTCCTTGAACAGTGCTTCTTTTTACTTGTTGAAAACTTGCTgtcctcctgtataagttagtatttgacatttctgtaagttagcttctgtcagtatttgacatatctgtaattagttcctcctgtgctttttcaataggactgaggtctggcaaactcggggagcagtgtgaggcagtcgtgcgctttcccaggcttttccagaagtacccgttccctatcctcatcaactcagcattcctaaagttagctgatgttttcagagttgggtaagtgtgctgaatgcctcagcagcccaaaggacaggctacttgtatctctgttttatactgtgtcttttcttctggtccattagaacttcttgccactcagttactctcggtgaatgcataatgctttgtaccatactttaatatgagatttaatgacacttttagaaaaaatgaaaaggttcagagcgaaattgtttcttcagggaacaagataaattaagtcagaacagccttattctgctactgtatatgtattttgtaaaattgtaggctttcttacagatgGTTTTAGAAATTCCTGATTTCATTTAGTGACTTGTAGGTACAGTCTTGGAGATGTGTATGCTCCTTACCCTTATCTCTTCTAAGCTGTGTGGAGGAAGATCGCCTGACTCACTGGCTGTCGCCT
This mutation is a 515 bp deletion beginning at Chromosome 1 position 191,582,302 bp and ending after 191,582,816 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40769 | CGT TCC CTA TCC TCA TCA ACT C | Wild type Forward | A | |||
40770 | TGG ACC AGA AGA AAA GAC ACA G | Wild type Reverse | A | |||
40771 | CCA GTT CCT TGA ACA GTG CTT | Mutant Forward | A | |||
40772 | CCT CCA CAC AGC TTA GAA GAG A | Mutant Reverse | A | |||
40773 | Fluorophore-1 | CAG AGT TGG GTA AGT GTG CTG A | Quencher-1 | WT Probe | ||
40774 | Fluorophore-2 | TGT TGA AAA CTT GCT GTT TTA GAA AT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40769 | 0.40 uM |
40770 | 0.40 uM |
40771 | 0.40 uM |
40772 | 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 |