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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= x bp
Wild Type = y bp
Wt Sequence (bp changes in brackets with wt first):
TGTGTCCCCACAGAACAAGAAAAAGAAGAAGACAGATTTCAT(c/a)C(c/t)TTACCGAGACTCCGTATTGACATGGCTTCTCCGGGAAAACCTCGGTAAGCATG
I304I, P305L
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39579 | TGT GTC CCC ACA GAA CAA GA | Forward | A | |||
39581 | Fluorophore-1 | TCA TCC CTT ACC GAG ACT CC | Quencher-1 | WT Probe | ||
40728 | CAT GCT TAC CGA GGT TTT CC | Reverse | A | |||
40729 | Fluorophore-2 | CAG ATT TCA TAC TTT ACC GAG ACT CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39579 | 0.40 uM |
40728 | 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 |