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.
Mut= 103 bp
Wt= 105 bp
Sequence (deletion in lower case):
AATCAGAGGCTTTCCCATGTGATAATGAAGTGTTTAATCACcataatgttatggagaaacagcatttaatgtttggctaaaccttctttctttatcttttcagataaattattagtcataactgtcgcaacaaaagaaaatgatggattccacagatttatgaattcagccaagtatttcaattacactgtgaaggtatgatatgtctttcagtctttggacttgttggaatatgtgcctaaaatatattaactagtggtttatttaaacatgttctataagcaacagaaatgtgtatatatgtaactttcctgacttcctttggagggtcatgtccacatacattgtaatcaaggtttgacggtaactattttcactgactaaagggatggcctgctgtggtgtcagttgaaaacatgatgggacggggcTTTGGGGGTGCGCTATTGACAGTGTTTTTCCTGTTTTGTTTGGTTTGGGTTCATTGGTTGGA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34387 | AAT CAG AGG CTT TCC CAT GT | Common | A | |||
34388 | Fluorophore-1 | TGG AGA AAC AGC ATT TAA TGT TTG G | Quencher-1 | WT Probe | ||
34389 | ATC TGA AAA GAT AAA GAA AGA AGG TTT | Wild type Reverse | A | |||
34390 | TCC AAC CAA TGA ACC CAA AC | Mutant Reverse | A | |||
34391 | Fluorophore-2 | TGC GCT ATT GAC AGT GTT TTT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34387 | 0.40 uM |
34389 | 0.40 uM |
34390 | 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 |