Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 129 bp
Wild Type = 131 bp
Wt Sequence (deletions in lower case):
AATGGGTCCTTCCACCTGCCTGGGAGACACTGTAATCCCCACTTGCATTATTATTGTAATTCCTACTTGCATATGAAGAAATGGTAATTAGAGGTGGAAGTGGCTTGCTTTCTCCTCTTCTGCCCAACCCCCTTTGTCCCTGTAACTCATTCACCTGGTGGAGGGAGTGGGTTTTGTAATTCACTTTTGGAGCTTGTGGGGGACAATGAGACCCATTGAAGGGCTTTTGTCTTGTTTTTCACCAGAACCTAGCTAGCATCCTCTTCAATTCCAACTAGAATGCTGTGGCAGGCACttcgaagatttggtcaaaagctggtgcggagacgtgtgttggagctgggtatgggtgagacccgccttgccagatgcctgagcaccctcgacttagtggctctgggtgtgggcagcacattaggtgcaggtgtgtatgtcctggctggagaggtggccaaagataaagcaggaccatccattgtgatctgctttttggtggctgctctgtcctctgtgttggctggactgtgttatgctgaatttggtgcccgggtccccggctctggttctgcatatctctacagttatgtcaccgtaggtgaactctgggccttcaccaccggctggaatctcattctctcctacgtcattggtgagatagatgcttggggtaggatgggagggatggatgattacacaaatagcagagggattatggatccagaaatccgagtgcaatTTCAGTGAAGTCTAAATTGTCTTTATTTTTCCTAGTATCTCAATACTTCTAGATGTCTGTTTGTTTGTTTATTTATTTATTTATTTGGGGTAGGGGTATAGGGAGGGTTTGAAGGTGCAGTACAATTGTTCACCTCCGTCTGGTGTTCATTCATTAAGATGTTAGTTAGGGTCTTTAAAATTAATAACAGGAGCTGGGTCTAGAGTTGTACACCTATAATTCCAGCACTCAGAGGCTAAGGTAGGAGGAT
This mutation is a 441 bp deletion beginning at Chromosome X position 101,083,784 bp and ending after 101,084,224 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48309 | CTT CAA TTC CAA CTA GAA TGC TG | Common | A | |||
48310 | CAC TAA GTC GAG GGT GCT CAG | Wild type Reverse | A | |||
48311 | CCC CTA CCC CAA ATA AAT AAA T | Mutant Reverse | A | |||
48312 | Fluorophore-1 | AGC TGG TGC GGA GAC GT | Quencher-1 | WT Probe | ||
48313 | Fluorophore-2 | AGG CAC TTC AGT GAA GTC TAA ATT GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48309 | 0.40 uM |
48310 | 0.40 uM |
48311 | 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 |