Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:44400086+44400175 90bp GCTCCGCACGATTTTGTTAG CCACCCAATCTCCATTACCA
Mut= 87 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence:
TGTCTGCTGCTTATCCACAATGAACCTGCTGATGTTGGACTCCGAAATCTGGACCCTGCCCCCTCCAAGGGCAAAAAGGgtaagcctctatcaagctgactcctagagcaccttcctttgcccctggctccgcacgattttgttaggactagcctttgccttgccctgtgcttgccactggatgtaggcgaaggaatggtaatggagattgggtgggagaagtaaagattctatcctgaaatcagccagcgcatcctgagccttctctgacagcacagatctctccattctgggtaaccccctaagtttaagcagtttgggtgccttcttccacctgcaccgtcctcatttcctttcatttgcatcgaggtaggggagagggaagcagggctttgagggggtgccccacatcaatcctg
Mutant Sequence:
CTGGCCCCCCTGTGGGAAGATCCTGAGGAAGGTGAGTGCATCTGATGAGCCTGAGGGGGAGGGCACCCAGTGGGCACTTGGGAGCACCTCTTCTTACACAACCCTTCCACCCCTAACCCATAGCAGGTTCCTTAGGAGTTCCCTGCCAGCTCCTGCTGCTGTTGGGATATTTTAGGAGCCTGAGatGTAGGCGAAGGAATGGTAATGGAGATTGGGTGGGAGAAGTAAAGATTCTATCCTGAAATCAGCCAGCGCATCCTGAGCCTTCTCTGACAGCACAGATCTCTCCATTCTGGGTAACCCCCTAAGTTTAAGCAGTTTGGGTGCCTTCTTCCACCTGCACCGTCCTCATTTCCTTTCATTTGCATCGAGGTAGGGGAGAGGGAAGCAGGGCTTTG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64037 | CCA CCC AAT CTC CAT TAC CA | Common | A | |||
64048 | GCT CCG CAC GAT TTT GTT AG | Wild type Forward | A | |||
64059 | TAG GAG TTC CCT GCC AGC TC | Mutant Reverse | A | |||
64060 | Fluorophore-1 | CTA GCC TTT GCC TTG CCC | Quencher-1 | WT Probe | ||
64061 | Fluorophore-2 | AGC CTG AGA TGT AGG CGA AGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64037 | 0.40 uM |
64048 | 0.40 uM |
64059 | 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 |