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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:104282410+104282510 101bp AATGGAAGACCATGCTGGAG TGGCCAGTTGTGTATGTCCA
Mut= 93 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence:
TCGCCAGGAGTTTGACACAATGGAAGACCATGCTGGAGATTACACTCTGCTCCAAGACCAAGAAGGAGACATGGACCATGGCTTAAAAGgtcagtggggtggacatacacaactggccagtcacagtg
Mutant Sequence:
ACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
12390 | GTC TTG TAG TTG CCG TCG TC | Mutant Reverse | A | |||
39873 | TGG CCA GTT GTG TAT GTC CA | Wild type Reverse | A | |||
52360 | CAT GAA GCA GCA CGA CTT CT | Mutant Forward | A | |||
52361 | Fluorophore-1 | AAG GAG ACA TGG ACC ATG GCT | Quencher-1 | WT Probe | ||
52362 | Fluorophore-2 | CGA AGG CTA CGT CCA GGA GC | Quencher-2 | MUT Probe | ||
oIMR7824 | AAT GGA AGA CCA TGC TGG AG | Wild type Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
12390 | 0.40 uM |
39873 | 0.40 uM |
52360 | 0.40 uM |
oIMR7824 | 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 |