Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:127567556+127567655 100bp AGATGGAAAGGCATTTTTGC GAAACTTCTATGGCAGGTGATTTC
Mut= 122 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TAGTATGCGAAAGCATATCCCTATTAAGATCAGTAAGAGAACTGGAGGGAGGGCTCAGCAGCTAAGAATTTCCTGCTCGTGCTGAGGAACCTGATTTCGGTAAAGCATCCCCTTCTGGTCTCTGTGGCACCCGACATGAATATAGTGCATATATGCATGTGTAGACAGATATGCGTGCCAAATAAAGAAATCTGTCTGTCTAGTGTATCTATTTGAGACAGGGCTTCTCCATGTAGCCTTGGCTGTCCTGAAACTTACTGTAAACTAGGTTGGCTTTGAATCTACAATCTATCTACCTCCACTTTCTGAGTACTGGGATTAAAGGCATGTACCCACATATGGTCATATTTTTTTTTTAAAAGTAATCAGTAATGTTTTCTAAATATATTGAATTTTTGCCATAAGAATGTACCAgcctatacagaatggtatactcatactctgtatagtatactatacggaatctttactcatactccagattaaatgattatcaagatattactttttaaagtgtattgcttcagttttagatggaaaggcatttttgcaaaataccgtgatgcatatgacaaatgaggctgtttgattctttcattgcagccaggaaatcacctgccatagaagtttcgcgtgctctgctagagtgtccttgtgagcatcgggcttctcagcaggtacgtatgtatctctctctaactactatgtttttcttgtgtagaaaatagtgacagattcatatgcatgtagcatatactttgattatactttctcactactctcccctgcccccattctggtctgtaTTCGTTTAAGCATCATTCGTGTCATACAGTTGATAAGAATTGTCTTGTTAGGGCTCCTGTTGCTATGATTGTTACTATGATGAAACACCATCACCAAAAGCAGCTTGGGGATCCGAGTGGTTATCTCAGCTTACAGTTCCAGGCAACAGTCCATTACTGGGGAAGTCAAGGCAGGAACTCAAGTCAGGCAATAGGGCAGGAATCTAGAGGCAGGAGATGGTGCAGAGACCATGGAGGGAGGAGAGCTGCTTACTGGCTTGCTTGATCTGCTTTCTTATAGAGCCCAGGACTATCAGACCAGGGACATGCCACCCACAATCCTCCCCCATCAATCACTAATTAAGAAATTGCCTTTCAGCCACATCTTTTTTTTCTTTCCTTTGACTTCCACATCATAGTCAATCATTGAAGGAAGTCAGGAACCTGGAGGCTGGAGCTGATA
This mutation is a 392 bp deletion beginning at Chromosome 3 position 127,567,439 bp and ending after 127,567,830 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48243 | AGA TGG AAA GGC ATT TTT GC | Wild type Forward | A | |||
48244 | GAA ACT TCT ATG GCA GGT GAT TTC | Wild type Reverse | A | |||
48245 | GGC ATG TAC CCA CAT ATG GTC | Mutant Forward | A | |||
48246 | AAC TGT ATG ACA CGA ATG ATG C | Mutant Reverse | A | |||
48247 | Fluorophore-1 | ATA TGA CAA ATG AGG CTG TTT GAT T | Quencher-1 | WT Probe | ||
48248 | Fluorophore-2 | TTG CCA TAA GAA TGT ACC ATT CG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48243 | 0.40 uM |
48244 | 0.40 uM |
48245 | 0.40 uM |
48246 | 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 |