Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:55251612-55251694 83bp TCTGGCACTGAATGGAAGAC TGAAAATGACCACTGCTGGA
Mut= 80 bp
Wt= 83 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
TGTTTAGCAAATTGTATCTTATATCTTGGGTATCCTAGGTTTGGGGCTAATATCCACTTATCAGTGAGTAGTTACTCTGGTTCTAAATGTTCTCGGTAATCTGATTTCAGCACTCAGTTCTTCTGTCATTCAGCAGGGGAAGGCTGGCGTGCGTATCCTTTGAACAGCAGGCTCAAGGAAACAGAGGTGACACAGAATGTGTCAAAAAGTCTCCTGTCCCATACTAAATCAATGACTACCACTCTTAATAACGTGCACACTGGATAAAGACTAGAAATGAGTTACACAAAGGAGATGATAAATAAAGGAGCGAACACTTAAGTCGTTCACCTGCcctctactgtgcttttatgacttacttctgcttttatgtgtttttctgcttcagcggaaagagacatcagtgtctactgtggagtgcaggccattacgatgaagattaatttttgcacggtccttttctcgggatattcagaaacagatctggcactgaatggaagacatggggattcccactgcaggggcttcatcaataacaacacctttccagcagtggtcattttcatcattaatctcagcaccttggagggctgtggaaacaatttagtggtaagatttctatgaaatcctgtgctatacctgaggtcataaataaacaagctccagtttcacgaaataatcaggagctcaatctatcttcaggaaaaaaaattccaataaagccaaacaaatcattttcctttgcttgtttaaaggttgaactcccaaatatacaattctgtcttctgataatggttctgttgataggagcTGGGCTGGAGAGATGCATGTGCTCCGctatggtcctCTAAGCAGCGTTCTGTTAGATTGACATTGATGGGCATCTCTAACAACGAGCCATATTCTTCATGCTTCTATGGGTACCTCAGATCATAAGAATGTTTTTGTTCCTTTGCTGTGATCTAGATCTTATGACCTCATTCAGTAAGAAAGATTGAAGATAAAATGGGAAACCTGAACTCGTTTTTCATGTTTACCCTTGACTGGTAGCCATCAATATTCTGTAAAAGAAAGCTGTATCTTGTGGGAAGTTCTTCATAGTGTAGGACTGCCCATTACATTTAAAAAATGCTTATTCTATCCAGTTTTATCCATAGTATTCTGGAAGTGCCCCTTTCCAGTTATTTT
This is a 477 bp deletion beginning at Chromosome 16 negative strand position 55,251,842 bp and ending after 55,251,366 bp (GRCm38/mm10). In addition there is a 10 bp deletion (CTATGGTCCT) 26 bp after the large deletion that will not alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37931 | TCT GGC ACT GAA TGG AAG AC | Wild type Forward | A | |||
37932 | TGA AAA TGA CCA CTG CTG GA | Wild type Reverse | A | |||
37933 | GGA GAT GAT AAA TAA AGG AGC GAA C | Mutant Forward | A | |||
37934 | GAT GCC CAT CAA TGT CAA TCT | Mutant Reverse | A | |||
37935 | Fluorophore-1 | TCC CAC TGC AGG GGC | Quencher-1 | WT Probe | ||
37936 | Fluorophore-2 | TGG GCT GGA GAG ATG CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37931 | 0.40 uM |
37932 | 0.40 uM |
37933 | 0.40 uM |
37934 | 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 |