Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 138 bp
Wild Type = 139 bp
chr10:79761705-79761843 139bp GCCTCTGCCTACCAGGAATC CCTCCAATGCAGTAACCAGA
The common primer (primer 59950) anneals over the nucleotide sequence containing mouse genomic variations rs240476437 and rs223341358 and the wt reverse primer (primer 59952) anneals over the nucleotide sequence containing mouse genomic variation rs222710171.
Wt Sequence (deletions in lower case): AGCACTCCCAATTCCGAATGGAAGTTGGATAGTTTAGGCTCCACACCCTTGGGTCATGCATGCTCCATGGCCTCTGCCTACCAGGAATCCCTGTGAAATAGTTGTACCCAATGgcttcctcaccattggtagaagagcacgtgggtggtggaaacagaacaatcactgccagtggaaggtcagcttggtctggttactgcattggaggccagtgtgggctgcagagcaagcctatcttgtgaggtggtctgcaggcccgtgacactgtctcttccacagGATCATCCAGCAGCTCGTGAATGGCATCATCTCTCCGGCTGCTGTGCCCAGCCTGGGCCTTGGTCCCTGgtgagtggggggcgggtgcccggcccctcctaccctgcgccccctgacccggcctctccccccagGGGCGTCCTGCACTCGAACCCAATGGACTACGCCTGGGGGGCCAACGGCCTGGACACCATCATCACGCAGgtacagcggccgctcaacatctgtggggggccgagaggctggcctagtaaagcagatggggtgctgctctgtagcaccagaggcctctgcagctatctcgttctctggtgtcacacagctctggccaagcccttcttcccctcacctcctgaagaatgcctgctgtcctagggtgcacagtggcctgtaccagagctgcacacccctgatacccctgAGGTTTAATGGGCCTGGTCTTGCTGATTTGCAACTCTGCAGAGCTGGGCAGGTGGAGACTCCCCACCCTACCACCCCATTGGGAACCATGCCCTCCCCAC
This is a 587 bp deletion beginning at Chromosome 10 position 79,597,047 bp and ending after 79,597,633 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59950 | GCC TCT GCC TAC CAG GAA TC | Common | A | |||
59951 | Fluorophore-1 | ACC ATT GGT AGA AGA GCA CGT G | Quencher-1 | WT Probe | ||
59952 | CCT CCA ATG CAG TAA CCA GA | Wild type Reverse | A | |||
59953 | Fluorophore-2 | TGT ACC CAA TGA GGT TTA ATG GG | Quencher-2 | MUT Probe | ||
59954 | AGG GCA TGG TTC CCA ATG | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59950 | 0.40 uM |
59952 | 0.40 uM |
59954 | 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 |