Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:23814789+23814898 110bp CGTTTTCAAATCAGGACGTT CTGGGTCTCTGTGCTCCACT
Mutant= 99 bp
Wild Type = 110 bp
Wt Sequence (deletions in lower case):
CCTAAATCAAACTAAGTATTTATAATAGAATTATAATAGATTTTCTTGAATTCAGAACAACTTTAGTTGTAAAGTCGTTTTCAAATCAGGACGTTTTAGCAAAGAGGgtgcccctactaactaagaaccctaaccctttctgtcttttccaggctgctgtctatcagtggagcacagagacccagggcattatctttagctccatcagctatggaatcatactgacgttaataccaagtggatatttagcggggatatttggagcaaagcagatccttggcgctgggttgctcatctcatccctgctcacgctgtttactccactggctgctgactttggggtgattttggtcattgtcattcgcacagtccaggggatggcacaggtatctcgctactattctcagaccgagtcgggtacagattcgcgacatctgcaaaacaacaaaggtctcaaccactttcattgcagggaatggcctggactggtcagttcacaatttgggcaaagtgggctccgccacttgaacggagcaagctcaccagcattgcgggttcaggtgaggacgtgtaagtgcgtcagtgggtggtgctttgctctgttcttctgtgacgcaggtgatttcaatattcaagaaaaaaatagagcaagatgtgacaattgcttaacaaaggagaaacatacatctgtgatgtagagcctcaggcttaggaggaaagcactgtgacctacatcagtcaaaggctGAGGCCTGATGGGAAAAAAAAAATACTGAGCTTTATGACAGGTAGAAATGGAATTAGTCATAGTGGCTATTGTCTGTCATCCTAATACTGAGGAGGCAGAGGCAGAAGGATTTCGAGTT
This is a 640 bp deletion beginning at Chromosome 13 position 23,814,821 bp and ending after 23,815,460 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46754 | CGT TTT CAA ATC AGG ACG TT | Common | A | |||
46755 | CTG GGT CTC TGT GCT CCA CT | Wild type Reverse | A | |||
46756 | GCC ACT ATG ACT AAT TCC ATT TCT ACC | Mutant Reverse | A | |||
46757 | Fluorophore-1 | CAA AGA GGG AGG CCT GAT G | Quencher-1 | MUT Probe | ||
46758 | Fluorophore-2 | TCT GTC TTT TCC AGG CTG CT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46754 | 0.40 uM |
46755 | 0.40 uM |
46756 | 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 |