Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 107 bp
Wild Type = 107 bp
chr7:16368430-16368536 107bp CGAATCTTGGAGAGCATGAA GTCATTGAGACAGCTCATTTCC
Wt Sequence (deletions in lower case):
TTGCTTGACTTTGTAGTTTAGATTACCTTGATAGCTCTGTCTTCATGAGAACTATGAGGTGTAGTTTATATGCTCATGGGTTAAGGAAACAGATTGTCTTGAGTGGTTTTACTGTCCAGCTAAATTCTTCTCTTAGGTGTCAGaagtggtcgtcatgtcgtgtatattttctacaccctgaaatctgcagcttttcttctgtcttttactcacagcacccaagaacctgttattcttgttgaaactggtcttaagcctgccagagagacagttaatttaagacttatgcagacagcagggcagtgattcacagtttgcagctcttagcagtcacttcctgtctcctctcaggtgtgcctgacttgctgctccagagatgtcatcattaaagtcgaccagatctgtcacagaaacagcattaagttcttcacaggagatgtctttgggtaccatgggtacacctttgcgaatcttggagagcatgaatttgtagagtaagtgctgggagagggccgaagggagcgttccacagttgAGTGGTATTTGTGGTAGGAAATGAGCTGTCTCAATGACATAGCATACTGTACTATAATACTTCAGAACATTCTCAGACAAGGGATTAACAAATTATGAAGC
This is a 392 bp deletion beginning at Chromosome 7 position 16,368,468 bp and ending after 16,368,859 bp (mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
60089 | CGA ATC TTG GAG AGC ATG AA | Wild type Forward | A | |||
60090 | GTC ATT GAG ACA GCT CAT TTC C | Common | A | |||
60091 | CAT GGG TTA AGG AAA CAG ATT G | Mutant Forward | A | |||
60092 | Fluorophore-1 | TAG GTG TCA GAG TGG TAT TTG TGG | Quencher-1 | MUT Probe | ||
60093 | Fluorophore-2 | TGT AGA GTA AGT GCT GGG AGA GG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
60089 | 0.40 uM |
60090 | 0.40 uM |
60091 | 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 |