Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 111 bp
Wild Type = 110 bp
>chr8:105355380+105355489 110bp CTGGGGAGGAAATGTCTGAG TGCAGACCTTGTACAGCACCT
Sequence (deleted region in lower case, ^x^ is an insertion)
CCCTGTGGCCGTGTTGGCCGTCTTTGAGGAGGTGCATGTCAATCAGACCCTCTTTATCATCATCTTCGGCGAGTCTCTGCTCAATGATGCAGTAACTGTGGTAAGAACATCTGGTGGCTTGCTGACCCCTGACCTTAGGCGTGACCGGGCTTAGACCTTTCCTCCTAGTCCCCTGGGGAGGAAATGTCTGAGCTTTCTTCTGTTCCCGCCCACTTCCCCC^ag^gtatggccccctcctacttcctgtctcccctcttgtcactcaggtgctgtacaaggtctgcaactcctttgtggagatgggctctgccaacgtgcaggccactgactacctaaagggcgtcggtcagtatttcccccaacttggcctgcattcaatgtctgccttctctggattgttgcgactcacccagccagcgtttgggacaGAGGAGGCTATTCCGATGTCTCATTCCTTCCTCTCCTCTATGGAGAAATGGGGTCTGGGAGGAGCTTGCCTTGGAATTCTAGTCTGGGATCCTGAGCCCCAGGGAGTGTGGTGGGTGCCTTCCTCTCTATTTCTTACTGGGCAACAGACTTTGTCAGCCCAGCTTGGGGAGGGTCTGGGCCAGGGGTCGTGCTGACAACCCACTCCTCCCCCAGCCTCCCTGTTTGTGGTCAGTCTGGGCGGGGCAGCGGTGGGCTTAGTCTTTGCCTTCCTCCTGGCCCTGACCACACGCTTCACCAAGCGGGTCCGCATCATCGAGCCGTTGCTGGTCTTCCTCCTCGCCTACGCAGCCTACCTCACTGCTG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37753 | CTG GGG AGG AAA TGT CTG AG | Common | A | |||
37754 | CTC CCA GAC CCC ATT TCT C | Mutant Reverse | A | |||
37755 | TGC AGA CCT TGT ACA GCA CCT | Wild type Reverse | A | |||
37756 | Fluorophore-1 | AGG CTA TTC CGA TGT CTC ATT C | Quencher-1 | MUT Probe | ||
37757 | Fluorophore-2 | CCT ACT TCC TGT CTC CCC TCT T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37753 | 0.40 uM |
37754 | 0.40 uM |
37755 | 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 |