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= 141 bp
Wild Type = 154 bp
>chr7:64578668-64578821 154bp GATCAAAAACTGTTCCCATTCT GCACTGTCCATGTGGAATGA
Wt Sequence: GTGTATGTCTCCTTAAATGAAAATGGTGACAAGAAGCCATGTTTTCATTTTTTTCTTTGGAGTTGGGAGCCACTAGAACTTTGCTTTGAAACCGTAGGCTTTTTATCTACCACTGATATTGCTCATCCCATGATTTAGCTTTCTAGCAAGTTTGACATTAAATTATTTGTGCAGTGTCTCATATTTTATCTCATTTCTTTGCCATCAAAATCAACTTAAGTAACATCGAAAATCtttatagtcatctgttactggcaatttgcagtggggtagctgtcttgtttcccaaaagctcaataaatacgagtcaggatctctcatcctttgaagttttaagaagacaagatgaatttagttttaaatgcaaattaagtccccaagagaaatctgcccacctgagcagcatgggcctgtgttgttagtggctgaggcttctagacactgattggtttgtgctcttcatgtttcactgtaccataaagtactaacagatatgtatttttcccccacagtaaaaccaactgtgatcgatgtggacatttatgttaacagcattggtcctgtgtcatccataaacatggtaagaaacacctgtgtttaccttggtagctcctgtgcgtcttcatgcttacagtctccactggatttctgttgcagttttattaagcagatttaaagcccacgactgactcaagtgttaagtgccgtggtattgaaaatagtctttaagaatcagaagggacagtgtgacatcagcagtgagtcattgctctgaagatcaaaaactgttcccattcttacatacatagatagcacatgccattcaaagggaattatttagggttgtgtactcatTGGCTTTAGGCAACCTCACTAATATTAGGGATGCACGTAAGTCCAGTGAGAGAAGTCATTCCACATGGACAGTGCTTAGCTGCTAAGCACGCGATGCTGTACAAGAATGCATTACACCAAGCCACCTGACATGTCTGCCAGCTGTGGTGTGCCTCCTGATTAGATGATGTAGAAGTCCCATAGCAGGTCATT
Mutant Sequence:tttatagtcatctgttactggcaatttgcagtggggtagctgtcttgtttcccaaaagctcaataaatacgagtcaggatctctcatcctttgaagttttaagaagacaagatgaatttagttttaaatgcaaattaagtccccaagagaaatctgcccacctgagcagcatgggcctgtgttgttagtggctgaggcttctagacactgattggtttgtgctcttcatgtttcactgtaccataaagtactaacagatatgtatttttcccccacagtaaaaccaactgtgatcgatgtggacatttatgttaacagcattggtcctgtgtcatccataaacatggtaagaaacacctgtgtttaccttggtagctcctgtgcgtcttcatgcttacagtctccactggatttctgttgcagttttattaagcagatttaaagcccacgactgactcaagtgttaagtgccgtggtattgaaaatagtctttaagaatcagaagggacagtgtgacatcagcagtgagtcattgctctgaagatcaaaaactgttcccattcttacatacatagatagcacatgccattcaaagggaattatttagggttgtgtactcat
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37071 | GCA CTG TCC ATG TGG AAT GA | Mutant Reverse | A | |||
37072 | GTG CAG TGT CTC ATA TTT TAT CTC A | Mutant Forward | A | |||
37073 | TCC CCC ACA GTA AAA CCA AC | Wild type Forward | A | |||
37074 | CAG TGG AGA CTG TAA GCA TGA AGA | Wild type Reverse | A | |||
37075 | Fluorophore-1 | AGG GAT GCA CGT AAG TCC AGT GA | Quencher-1 | MUT Probe | ||
37076 | Fluorophore-2 | TGG TCC TGT GTC ATC CAT AAA CAT G | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37071 | 0.40 uM |
37072 | 0.40 uM |
37073 | 0.40 uM |
37074 | 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 |