Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:126940894-126940992 99bp TGATTGGATTTTCCTTTCTTCC TGGTTCTTTTGCTACTGTGCTG
Mut= 90 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGGAATCTGTATATTTCAGTAATAATGGCTTAATTATTTTATTTAGAAAGGGGGCTTTGTTCTGCTGCTGCACAGCTGTTGGGACAGTCTCTGTCTTCTGAATTGTTGGACCTGGGGATGCAGAAGAAAAGAGAAAGATCCTACCTAGAAACACCAGTTCACTCGCCAGGGAGgcagggattctggaggcaggaacgttttgtagaattaatgatggttgcagcaattagatgtcttgatccttaatgatgatagccaagtttttaatttatttttcttgtaggaagaaagaaacagaagcttgctaaggagagagctgggctttccaaggtaatgaaaatagtcttgtgtttttgcgtgtagattgcatgctctctaagcctcctgtgttttcctttggtggtggtaagagcaaatatgttaattgggtaagcagcaagatgagagtctccagagatggacatgaagagttaaagttctcggctacttaaaaatggttttaaaaaatacagcatttgattggattttcctttcttccttccgacagtaacctttaatcctagtttgagAAGCTCTGTTAGAGGCAGCCTGGGCAGCACAGTAGCAAAAGAACCACGTGTGTTAGTCCCACAGATCTTTAAGGTTTTTACACCTATGGATCAGAATTATATCAGGTTCTGACATTTAAAAAAACAAATTGTAATTTGTAGAATATAAATTTTCATTTTTTTCTTTTCATAGGTCCTTAGTTTACAATTTTTAATGACTGTAGGTTTTTATCTACTTAATT
This mutation is a 396 bp deletion beginning at Chromosome 8 position 126,940,940 bp and ending after 126,941,335 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38903 | TGA TTG GAT TTT CCT TTC TTC C | Wild type Forward | A | |||
38904 | TGG TTC TTT TGC TAC TGT GCT G | Common | A | |||
38905 | AGA GAA AGA TCC TAC CTA GAA ACA CCA | Mutant Forward | A | |||
38906 | Fluorophore-1 | TCC GAC AGT AAC CTT TAA TCC TAG TT | Quencher-1 | WT Probe | ||
38907 | Fluorophore-2 | ACT CGC CAG GGA GAA GCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38903 | 0.40 uM |
38904 | 0.40 uM |
38905 | 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 |