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= 112 bp
Wild Type = 110 bp
Wt Sequence:
cctattgccctcaaagaccataaggaggctttggatcccctggaactggagttaaaaacagttgtgagctgaaatgtgggtactgggaataaaacacagatcttctggaagag
agccagtgctcttaaccattgagctatcttttccagcctgattTTTAtcttctatggtaagcattttggtatttatcctaagcacagtgagaattttaagcaagggaagtgatatcacttttcta
Mutant Sequence:
CTTTCCCGCCCTCCCTTGCGCTACGGGGCCGCCCGCACCGGCCTACACGGAGCGCGCGCGGCGGAGTTGTTGACCT
CGAGCggtaTCTTCTATGGTAAGCATTTTGGTATTTATCCTAAGCACAGTGAGAATTTTAAGCAAGGGAAGTGATATCACTTTTCTA
TGTTAGAAATATTACTCTGGAATCTACTACATAGAATGAGTAGTACTTTGGCATGCAAGTTGATAAGAGAGAACTTACACTAATACAATTAATGCCCTCCTGGGAACTCTAGGAAAGAGATATGCACTCG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
29854 | CCC TTG CTT AAA ATT CTC ACT G | Common | A | |||
29855 | CCG CAC CGG CCT ACA | Mutant Forward | A | |||
29856 | GAG CAG CCA GTG CTC TTA ACC | Wild type Forward | A | |||
29857 | Fluorophore-1 | TGA GCT ATC TTT TCC AGC CTG A | Quencher-1 | WT Probe | ||
29858 | Fluorophore-2 | CGG CGG AGT TGT TGA CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
29854 | 0.40 uM |
29855 | 0.40 uM |
29856 | 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 |