Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:5160196-5160313 118bp TGGGCAGATAACAGAGAAAATG TGGTTGTAAGAGTGATTGGTCAG
Mutant= 123 bp
Wild Type = 118 bp
Wt Sequence:
ggtccacccttctcccttcatccctcttagaacatctctttcgtcataatcctggtgactgtgttgaatcacatagagaaaacatctgggcagataacagagaaaatgatgtttctaagcttatgtagacctaccagcaccctgcagtaccaggccctctggcatttctttctttctttctctgaccaatcactcttacaaccaagagacgctaaagccactaacactcagtcctctagc
Mutant Sequence:
aaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatccccatcaagctgatccggaacccttaatataacttcgtatagcatacattatacgaagttatTCTGGCATTTCTTTCTTTCTTTCTCTGACCAATCACTCTTACAACCAAGAGACGCTAAAGCCACTAACACTCAGTCCTCTAGCTGGACCTGAACCACCAAGGTTCTGGTTGCT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
25183 | ATC ATG TCT GGA TCC CCA TC | Mutant Forward | A | |||
51677 | Fluorophore-1 | AAG CTG ATC CGG AAC CCT TAA | Quencher-1 | MUT Probe | ||
63392 | TGG GCA GAT AAC AGA GAA AAT G | Wild type Forward | A | |||
63393 | TGG TTG TAA GAG TGA TTG GTC AG | Common | A | |||
63394 | Fluorophore-2 | CCA GCA CCC TGC AGT ACC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
25183 | 0.40 uM |
63392 | 0.40 uM |
63393 | 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 |