Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:26592461+26592569 109bp TCTCTTGAATCTTGTGCATTGA TGAAGTGCTGAGCTAAAACTGA
Mut= 93 bp
Wt= 109 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GAGTTTCACCATGTTTTCTGGGCTGGCCTTGAACTATGGGACTCAAGGTAGTCTCTTGATATAGCTTTACAAGTATCTGTGACTACAGATGACACCACAGCCTTAACTATGATTTCTAAAAAACTTTTTGGCCATAGccaactagaattttcggcatacccactatatgctaacgtaacttaacttctttcttccaggtgatatatcctcagcattccaaacttactgataaagaagtaagtaagcacgtctgtctgtctgtctgtctgtctgtctgtctgtctgctgtctgtcacctatgctcgttcctcgtgcagtgtgtgtgtgccggcaccgacggctctgcttttctctttcagaaaaccaatatttgctacttgtcttttccagactcaaattcaggtaactttttatcataagatgtttctcttgaatcttgtgcattgaatatcatatggtttgtagttttcgtacatgacgagtagccagCACGGCATGTTAAGATGGCATGCTCAGTTTTAGCTCAGCACTTCATTGATTCTGAGTTTTCTTAATGACGCCTAGTCCTGTCCTTACTGATTTCACTTAAGGTCACTGTTACAGTGTGATAAGGTAAAATATAAAAAGTACAATACATTC
This mitation is a 352 bp deletion beginning at Chromosome 14 position 26,592,173 bp and ending after 26,592,524 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43406 | TCT CTT GAA TCT TGT GCA TTG A | Wild type Forward | A | |||
43407 | TGA AGT GCT GAG CTA AAA CTG A | Common | A | |||
43408 | TGA CAC CAC AGC CTT AAC TAT G | Mutant Forward | A | |||
43409 | Fluorophore-1 | TTC GTA CAT GAC GAG TAG CCA G | Quencher-1 | WT Probe | ||
43410 | Fluorophore-2 | TTG GCC ATA GCA CGG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43406 | 0.40 uM |
43407 | 0.40 uM |
43408 | 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 |