Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:72384915-72385021 107bp GGGCTGTATGGTCCTGACAC CCACTGGTGGCTGAAACAG
Mut= 111 bp
Wt= 107 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATCCTGAGACTATCTCCATATGCTATTGTTCTGGGTTTACAGGAAGCAGAAATGGTAGCAGCAGAGATCCCCCACTGACCCGAACTTCACTGTATGTGTGTATGTTTATCTGACCTTTTTTTATTCTCTCTGCTCCTAGCCAGGGTCCTGGACTGGGCTGTATGGTCCTGACACTGTCAGTGAGAAGTATAAGCAGAGctgagactgggacagggctcacaaataagtcactctgttgtaacctgtttcagccaccagtggcctgggttgtgcctgtggcagataagatgcgctttgatgagatctttttgaagacagacctggaccttgatggctatgtgagtggccaggaagttaaggagatcttcatgcactcaggcctcactcagaaccttctggcacacatctggtaatgatggaaacattggtggaggtgctctgagaggctgggaaagagggtggggagggctcccgttttccaggtgacagtatacagggagGTAGGGTCAGCCCTTATCTGATACTGGAGAGCTGTGTTGAAAATAGTCTTCTCCACAAGCCTTACCTTGTCACAGCCACCTGTAACGGTTACTCTGCCTGCCACTTGAGTAACCTCAACAAGGACCTTGACAGTTCCAGTGAGGTCCCACTTGGACATGGGCATTCCAAGTACATTGCACTGAAGTCGAGTGGGTCACCAGAGTGTGATTTTGTGGGGGGCAA
This mutation is a 302 bp deletion beginning at Chromosome 8 position 72,384,676 bp and ending after 72,384,977 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39994 | GGG CTG TAT GGT CCT GAC AC | Common | A | |||
39995 | CCA CTG GTG GCT GAA ACA G | Wild type Reverse | A | |||
39996 | AGG TAA GGC TTG TGG AGA AGA | Mutant Reverse | A | |||
39997 | Fluorophore-1 | CAG GGC TCA CAA ATA AGT CAC TC | Quencher-1 | WT Probe | ||
39998 | Fluorophore-2 | AAG CAG AGG TAG GGT CAG CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39994 | 0.40 uM |
39995 | 0.40 uM |
39996 | 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 |