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= 175 bp
Wild Type = 178 bp
This assay is capable of distinguishing hemi from hom.
Wt Sequence:
ccagggcaaaacctcaaaactctgtactggacatttctcaaggccaggtcaatgcaaacatcacatgcacacggtttggaagtgctgccctacaagggctCTGActcaacccattttagacataatagttgactttgaccttcaaattgacagg
tcctatgtgaaacataaagattttggaatttcagaagattaacaaacgtagcatacagaacaaaatatggtgcacgatcagcatcaccctgttctgagtcaagatttttg
Mutant Sequence: ATCAAAGAAGTAAGCTCCACCTTAGAAAAAAGTGGAACGTCATGCTAaggACTCAACCCATTTTAGACATAATAGTTGACTTTGACCTTCAAATTGACAGGTCC
TATGTGAAACATAAAGATTTTGGAATTTCAGAAGATTAACAAACGTAGCATACAGAACAAAATATGGTGCACGATCAGCATCACCCTGTTCTGAGTCAAGATTTTTGTTTTGTTTTGTT
TTGTTTTgttttgttttgttttgttttgttttgttttatatccatggctggcaggcatattag
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
20239 | TGC AAA CAT CAC ATG CAC AC | Wild type Forward | A | |||
28843 | CGT GCA CCA TAT TTT GTT CTG T | Common | A | |||
28844 | CAA AGA AGT AAG CTC CAC CTT AGA | Mutant Forward | A | |||
28845 | Fluorophore-1 | CCT ACA AGG GCT CTG ACT CAA CC | Quencher-1 | WT Probe | ||
28846 | Fluorophore-2 | AAA GTG GAA CGT CAT GCT AAG GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
20239 | 0.40 uM |
28843 | 0.40 uM |
28844 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |