Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:6619969-6620077 109bp AAAAGACCCCCATCAACCAG GGAAAGCCTATATCCTTGTTTG
Mut = 111 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AGCATCAGCTCAGTTGCTAGCTGATATCGGTGGCGTAACATCTTTGAGGGGCATCTAGAAAGATGAGAAAACATTTTCCTAGCAGTTCTTCCTGTCAgccccagccttttgcccagaactcaattccatctggtcctctgaaatatgttcaccttctgccttctcagcctgcctacgttcaggcttgtcaccaggccaaccctctttctgctttctgtcctatctcttttccccctctgaagaatacttcattctatcctaccttaatgatttccatgccctgctcttcctgccttactaaactttccttgaagcaccatattcccttcgaacctcccgcccattttccctctaccccagctacccttccttctaataaaagacccccatcaaccagttccgtagtctttcaccctgatcagaaaactagccactctcaggttgaTAGGGTAAGTCTAAAATCTCAAACAAGGATATAGGCTTTCCTGTAAATGGATAATTTCAGAAATGCACTTAGTAAGAGTTGCTAGAAACCCTGTGCTGTGAACCCAGAAAGAGCAGT
This mutation is a 348 bp deletion beginning at Chromosome 3 position 6,620,010 bp and ending after 6,620,357 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51085 | GGA AAG CCT ATA TCC TTG TTT G | Common | A | |||
51086 | AAA AGA CCC CCA TCA ACC AG | Wild type Forward | A | |||
51087 | CGG TGG CGT AAC ATC TTT G | Mutant Forward | A | |||
51088 | Fluorophore-1 | ACC CTG ATC AGA AAA CTA GCC AC | Quencher-1 | WT Probe | ||
51089 | Fluorophore-2 | CCT AGC AGT TCT TCC TGT CAT AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51085 | 0.40 uM |
51086 | 0.40 uM |
51087 | 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 |