Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 119 bp
Wild Type = 131 bp
>chr11:99486755-99486885 131bp TGTCTGTCCTGAAAGTCACTGG GGGCATTGGCCATCTTAC
Wt Sequence (deleted region in lower case):
GTCTGATTTCCAGTGGCTCTTGAACATATATGGTGCGCTAAAGTCATGCATGCAGACACACACACACACACACACACACACACACACACACACACACAAAACCTTTTTGTAAAAGGAGAATAGTTTGTCTGTCCTGAAAGTCACTGGTGGAAGAAACCTGGATGCAGACACactgctgtcatggctggatctgtccaactggaataattccgtgaacatattttacagatagtggacggtaagatggccaatgcccacattgtcgtgctcattgacaacgccaggatggcagtggatgacttcaacctgaagtaagtcccttcctcacagagttccacagaggcaaagctcggcctgccataacctgctgccaaaatcttgctggattataactaaagggcgggacaaaagtcacatgcaatttaagtaccccaggtacacgtgcacGTGATAGCATGTGAAGATTTTAATACAAACCAAGAATTGGGTCGTATTAAGAACTTTCCACTTCAGGTACAGTAATATTGTCATACTTACTTAGAAAAAAAATTTTTTTCCTTAAAGAAAATGTACTT
276 bp deletion beginning at Chromosome 11 positive strand position 99,486,564 bp and ending after 99,486,839 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37241 | TGT CTG TCC TGA AAG TCA CTG G | Common | A | |||
37242 | ACT GTA CCT GAA GTG GAA AGT TCT | Mutant Reverse | A | |||
37243 | GGG CAT TGG CCA TCT TAC | Wild type Reverse | A | |||
37244 | Fluorophore-1 | CAA ACC AAG AAT TGG GTC GT | Quencher-1 | MUT Probe | ||
37245 | Fluorophore-2 | CTG TCA TGG CTG GAT CTG TC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37241 | 0.40 uM |
37242 | 0.40 uM |
37243 | 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 |