Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:136229606+136229692 87bp AGGGAGCTGTTGTGAAAGGA GCTGCAATACCCTTGTCCAC
Mut= 104 bp
Wt= 87 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACTGCACCTGACTGGTTCTTGGCAACCCTGGGGAGAAGCTGGAGTGGGCTGAGCTTTGGGAAGGGAGTTGGGTAGGTGTGTTAGTGTCAGCTGGACAACAAGACTCAAGAGTGAGGTTCCCCCTGAAGGCAGCAGAGTGTCCTGTTTATCCCAGGGCCCctgctaccctttgggcaactcattcttttctcaactccaggattccaaaaagcatctggagaaggcgtggaaagactacgaaagcaaagtgtgagtgaccttggacgggtggccttcaggagagcacgtctgctctacattgaagaaggaggctgattttgggatgtgtcacttggggtgtctggctctttggagcctattgtcaggcctggctgggttctgggggccatgttgcaagatgaccaaagggagctgttgtgaaaggaccaggggatgctgcccCCTGGCCACCTTCTGGCCTTGTGATGTCCATGTGGACAAGGGTATTGCAGCCTCAAGATAACACAGCCCTGGGGCTGGAGAGATGGCTCAGTGGTTAAGAGCACCAACTGCTCTTCTGAAGGTCCTGAGTTCAAATCCC
This mutation is a 282 bp deletion beginning at Chromosome 4 position 136,229,360 bp and ending after 136,229,641 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42909 | AGG GAG CTG TTG TGA AAG GA | Wild type Forward | A | |||
42910 | GCT GCA ATA CCC TTG TCC AC | Common | A | |||
42911 | AAG AGT GAG GTT CCC CCT GA | Mutant Forward | A | |||
42912 | Fluorophore-1 | CCA GGG GAT GCT GCC | Quencher-1 | WT Probe | ||
42913 | Fluorophore-2 | TTT ATC CCA GGG CCC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42909 | 0.40 uM |
42910 | 0.40 uM |
42911 | 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 |