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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut=112 bp
Wt=112 bp
Wt Sequence (bp changes in brackets with wt first):
CATGCCCACAGCATCACTACACTCAACCATCCTTTGAACTTGACCAAGGAGCAGCTATCTGAAG(c/g)TACACTGTGAGTCCATCCATTTACTATCAGTGGTCATGTTGCTCAGG
A1541G GCT>GGT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39014 | CAT GCC CAC AGC ATC ACT AC | Forward | A | |||
39015 | CCT GAG CAA CAT GAC CAC TG | Reverse | A | |||
39016 | Fluorophore-1 | AGG TAC ACT GTG AGT CCA TCC A | Quencher-1 | MUT Probe | ||
39018 | Fluorophore-2 | TGA AGC TAC ACT GTG AGT CCA TC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39014 | 0.40 uM |
39015 | 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 |