Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 97 bp
Wild Type = 106 bp
>chr6:38041123-38041228 106bp CCATGGTCCTCAAGGGTTC ATGGTCTCCACCGCATCCT
Wt Sequence (deleted region in lower case)
GACTATTTGCCTAACCTGCACAAAGCCCTACATTTGATTCTCAGTACCACATAAACTGGGTACAGAGGTACCTGTCTGTAATCCCGTGTGAGAGGTAGAAGCCTGAGAGTCAGGAGTTCAAGGTCATCCTCAGCTACGTAGGCGTTTGAAGTCAGTCTAGGCTACACAAGACGGAATAAGAAATAATAATGTTGCATATACCTGTAGAGGTGGGCCATGGTCCTCAAGGGTTCCCCTGCCCCCACACCAAGTGgttctcatactctgccttccttgtcagagaccaagacattcacggtggaggatgcggtggagaccatcggcttcgggcgcttccacattgcactcttcctaatcatgggcagcaccggggtgagcacacttggagaagctctgtggctgtgattctgtcctcactgtttagcaccaagttcctgctgacccccagcactggactgcccattcccgggagtaggaatcctgtggaaattatttcatcagcctttgcagtgggttggctgggaacgtgtctgttctggtaattcaagactccatcttagaggtttacactgagctcttgggaagaagggaaaggactgtggggttactccacgtggagtagctgcaagCTCTGAGCATGTGTGCATTGGTAACACCTGGAGCACCTGCTGTCGGGAAGACTCTAGATCCCCTAAGAGACTCAGTCATTTCCTTCATATCAGTGGTATTTGTTTCTCCAAAATCCTTGGTGTTGTGTGTTAGGCATGGTGGGTGCATCTGTAGTCACTGTATTTGGCAGATAGCTATAAGAGAATCAGGAGTTTAAGGTAATCTTCTGCTAGGTGTTGAGTTCGAGGCCAGCTAGGGATATGTGAGATCTAACTTTCCTCCAGATGTACACAGT
376 bp deletion beginning at Chromosome 6 negative strand position 38041189 bp and ending after 38040814 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38035 | CCA TGG TCC TCA AGG GTT C | Common | A | |||
38036 | TCT AGA GTC TTC CCG ACA GCA | Mutant Reverse | A | |||
38037 | ATG GTC TCC ACC GCA TCC T | Wild type Reverse | A | |||
38038 | Fluorophore-1 | TGA GCA TGT GTG CAT TGG T | Quencher-1 | MUT Probe | ||
38039 | Fluorophore-2 | TCT GCC TTC CTT GTC AGA GAC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38035 | 0.40 uM |
38036 | 0.40 uM |
38037 | 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 |