Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 99 bp
Wild Type = 116 bp
>chr6:128366298-128366413 116bp ATGGAGGTCACATGACAAGC CGACCCCAACACAAGTCAAC
Wt Sequence (deleted region in lower case)
CCTGGTTGGCCTGGAATTCACACCAGTCCTTCTGCCTCGGCCTTATGAGTGCTAGAGGGGTATGGGTGTGCACCATACATTGGAAAAAATTTTCTTTTTTAATTTCTTTCCCTAAATCTGAGATTTTTACCTCCTTAAGTATCCACTTTgCTATATGGACAAACCAAACTGTACAGCTCAGTGTGTCTGTTTATTAATAAAGACAATTTATGGAGGTCACATGACAAGCTTTCATGAATGCTTTGGAAGTTTCGAGATgaatgttctctcttggtacctctctcaggttccctttctctcttgcagttgacttgtgttggggtcggagatgtctgtaataaagggaaggtaagcacgtgggtgagggtagagctccaggcccttgcctccattgtgactcccagggactcccagggaacgtcagcctctgataagctctccccaaccctcgtgtctctcctgtgcctctctgtctgtctctgtctgtctgtctgtctgtctgtctccctctctctctttctctctctctctctctcccagaacctggtggtagcagtcagcgcagaaggctggctgcacttgtttgacctgacacccaccaaggctctggatgcctctgggcaccatgagacacttggagaggagcagcgacctgtcttcaagcagcacatcccggccaacacaaaggtcatgctgatcagcgacatcggtgggcatgagcagccaaggggcgggggccagtgggttcctgggcagaccccagggGACTGGGCCAAGATGGAGAGTTGTTGCCATAGCAACCCTAGGTGTGGAGAGCTGGCACCTTGgtGCACATGTGCAGTGTTTACCTTCTCTCTTTGAATTTTGTTTTACTTTCTCCTCATTTGTTGTATGTTTAATGTATAGCTACTGAGCTCCTGTTACAAGCCAGGAACTGTTCTAGGTTCTAGAATTACAAAAATGAATGAAGCAAGCAAAAAAAAAAAAAAAAATCATGACCTCTAAGAATAGACAGAA
507 bp deletion beginning at Chromosome 6 negative strand position 128,416,346 bp and ending after 128,415,840 bp (GRCm38/mm10). In addition, there are 2 small intronic indels, a 1 bp (G) deletion 108 bp before the exon deletion and a 2 bp deletion (GT) 62 bp after the deletion
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38329 | ATG GAG GTC ACA TGA CAA GC | Common | A | |||
38330 | TCT CCA CAC CTA GGG TTG CT | Mutant Reverse | A | |||
38331 | CGA CCC CAA CAC AAG TCA AC | Wild type Reverse | A | |||
38332 | Fluorophore-1 | TGG GCC AAG ATG GAG AGT T | Quencher-1 | MUT Probe | ||
38333 | Fluorophore-2 | TGG TAC CTC TCT CAG GTT CCC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38329 | 0.40 uM |
38330 | 0.40 uM |
38331 | 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 |