Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:11162786-11162895 110bp CGGAAACGCACAGTTTAAGA CCTCCACTTCCCAAGTTCTG
Mut= 101 bp
Wt= 110bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTGAACTCAGGACCTTTAGAAGAGCAGTCAGTGCTCTTACCCACTGAGCCATCTTGCCAGCCCGAGTGTTGGATCTTTGAGGTGCCATGCGGCTTCTGGGAACTAAGTTCAGAACACCAAGCATTCCTAACTACTCAGCCATCTCTCCAGCCCCGGAAACGCACAGTTTAAGATAACTTCTGGCtgaattatgctgatagagggcctgtcatggtacagcatggtagagcgttcttgtaatctcagaacttgggaagtggaggcaagctgatctcagtaagttccaggccaatcagggttgctgccttagaactaacaaaacttttagtagaacttggtaattacaggagatttgcttcttcctctcaggcctccgctgtcatatcctgggacccaggagcagtatgcggtaaggatgtggggagccctatggctgggcacagggcctcaagagctgtaatctgcagcaggatttggcagtctagccttgactctcacacctgaccagctgccctccttcataaagagagctttactggaacaccgcggccacaggcggttgttcatgggttctctgtggtgcctgtgtgagacagggcagaactcaaatgtcccacagtgctttaaatattctctttggcatttacagaaaaagcttgccaatctcttctatatagtgtaccctttcggggcatttctggacAGTTGTCCTGCATTTTCTTTCCTGTGTTAGTGTAGTTTGAATTAGTATGCATTTTTAGTCCTTTGCCAGTTAACTAACATTTTATCAAAAATAGGTGGGATAGAATTCAGCAGAGAATGTGGGGTATCATTATTTTTGTTTTTTAAGTCCTGAAAGCAGTGAAGGAGGATTGTCTACATAGGGAG
This mutatuion is a 519 bp deletion beginning at Chromosome 16 position 11,162,346 bp and ending after 11,162,864 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41667 | CGG AAA CGC ACA GTT TAA GA | Common | A | |||
41668 | CCT CCA CTT CCC AAG TTC TG | Wild type Reverse | A | |||
41669 | ACT GGC AAA GGA CTA AAA ATG C | Mutant Reverse | A | |||
41670 | Fluorophore-1 | TGA TAG AGG GCC TGT CAT GG | Quencher-1 | WT Probe | ||
41671 | Fluorophore-2 | CTT CTG GCA GTT GTC CTG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41667 | 0.40 uM |
41668 | 0.40 uM |
41669 | 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 |