Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
Mutant= 105 bp
Wild Type = 136 bp
>chr2:121976707+121976842 136bp GAAATCCAAATGCTGAAGAACG TGCAGGCGTATGTATCAGTCTC
Wt Sequence: AAACCCCTCAAATTCAAGTATACTCACGCCACCCACCGGAGAATGGGAAGCCGAACATACTGAACTGCTACGTAACACAGTTCCACCCGCCTCACATTGAAATCCAAATGCTGAAGAACGGGAAAAAAATTCCTAAAGTAGAGATGTCAGATATGTCCTTCAGCAAGGACTGGTCTTTCTATATCCTGGCTCACActGAATTCACCCCCACTGAGACTGATACATACGCCTGCAGAGTTAAGCATGCCAGTATGGCCGAGCCCAAGACCGTCTACTGGGgtaagcctcaagttcttccttactttctggacgctccatctgtgtggacttaaaactgctttgctattttaaaaacctgcatactgagattgttagagaataccc
Mutant Sequence:CCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGGATCggcaataaaaagacagaataaaacgcacgggtgttgggtcgtttgttcggatccatcgaTGAATTCACCCCCactgagactgatacatacgcctgcagagttaagcatgccagtatggccgagcccaag
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35415 | TGC AGG CGT ATG TAT CAG TCT C | Common | A | |||
35416 | GAA ATC CAA ATG CTG AAG AAC G | Wild type Forward | A | |||
35417 | Fluorophore-1 | TCG GAT CCA TCG ATG AAT TCA C | Quencher-1 | MUT Probe | ||
35418 | Fluorophore-2 | CAG ATA TGT CCT TCA GCA AGG ACT G | Quencher-2 | WT Probe | ||
oIMR8394 | AGG GGA TCG GCA ATA AAA AG | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35415 | 0.40 uM |
35416 | 0.40 uM |
oIMR8394 | 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 |