Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:83934941+83935061 121bp TACAACAGGACTGGATTCAGCA AGTATCTAACATATTATCACGCTTGT
Mut= 136 bp
Wt= 121 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATGTTTTTATATTTGAGGTGTTATTTTTTTAATTTATGCTTTTCTTCAGGTCTAGATATATGTGCATTTCAAAATGAATGTTTTGATTCTTGATTCAAGAAATATTTATGCTAAATATTTTTTAAGTGGAATTAATATGGTATGGGGTTAAATGTATTTGTTTGGAGGACTTTAATTTTTGTTTGTATAAATTCTGTTATATCTAAATGCTATTTCAGATTAGATTTGTAAATTTCTCAAATATTCTTTGTTATTTAAAAAAAGCCTAATtgatttgtcagcttgagaattgttttaattcaaccttctttttttacaacaggactggattcagcatcaaaatacctccacccacattgagagctgtcggcagttaaggcaacagtaagaatagtttcttcccttcTTTACAAGCGTGATAATATGTTAGATACTTTAAAGCTATTGGTTATTTAAAATAATAATCTTAGAAAACTATGTTTAGTTTTCCAAAAGGAGAGCTTCATGTGATCTTTCACTGACTCCTTCTTAACTTAATCTCTAGTAGGTAATTATAGACTTTTTTTTTTTTTTTTTGTGTCTCGTTTGCCTTTTCCAGAATGGTTGAGGACAGATAGCATTCTTTTCTATTATCTTGGGCCATTTCTCAAAGGTTGCATAATTAAAAACTAACTCAGACCATATGCCAGCATTTAATTTCCATTATAAAGACTTTCC
This mutation is a 136 bp deletion beginning at Chromosome 6 position 83,934,897 bp and ending after 83,935,032 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47486 | TAC AAC AGG ACT GGA TTC AGC A | Wild type Forward | A | |||
47487 | AGT ATC TAA CAT ATT ATC ACG CTT GT | Common | A | |||
47488 | GGA GGA CTT TAA TTT TTG TTT GT | Mutant Forward | A | |||
47492 | Fluorophore-1 | ATG CTA TTT CAG ATT AGA TTT GTA AAT TTC TC | Quencher-1 | MUT Probe | ||
49149 | Fluorophore-2 | TGT CGG CAG TTA AGG CAA C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47486 | 0.40 uM |
47487 | 0.40 uM |
47488 | 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 |