Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 150 bp
Wild Type = 158 bp
>chr13:64799187-64799344 158bp GCAAAACTATTTGAAAGAAGACAGC TGTCTGCATTTGTGTTTCCTG
Sequence (deleted region in lowercase, indels in parentheses)
GACAAAATTGGTGCATACGTGCTATGGGATGCCACAGTACCAGGAGACTAAGTTTCCGTGAAACTGTTCACCTCGGGACTGTGTCCAGGTTCCTAAATCTGTCAAGCAAGTCATTACTGGAGTGGATGTAGCAAATAGTAGATAATTGAGAAAGCAAAACAAAATTGCTATATCTATGTTAAACATTAGCAAAACTATTTGAAAGAAGACAGCCTTTT(ggata)TAGGCAGAGTGAACACATGCCACCATTGAtataagctatgataatacaaaaatgatcttgaagatacgttgacccttcctctttgttttttacagggatttccaggaaacacaaatgcagacagtgtggtacaatatagtctccagccttccttccacaccaggttcctgcgcttcctccccttaacctggaaccctaaaggcaggattgggatgcggatagaagtatatgggtgttcatacagtaagtgatatttattttccatgtaaaataaacatctgcatgagatgctgcaaaagccagcgtcAGTTAAAAATTACTTGCTCTAGACATTTTTAGTAATTGTATAATTAAAACCGA(gt)GTTATGAGACTTCTACTAACTCCTTATGACAATATAGGCTTTAATTATTTTTTTCATAGTTAAATTTAGAGATTGCTTAGTGAGAAAAATGTTTGATAGGAAATAAGGGTGTAATTTTGGAGCCCTAGTATCACATAAAAGGGCAGGTGTACAGTGAATGTCTACGAACCCAGTGCTGGAGACAGAGAACAGAGACAGGTAAATCT
278 bp deletion beginning at Chromosome 13 negative strand position 64,799,280 bp and ending after 64,799,003 bp (GRCm38/mm10). There are 2 additional small deletions, a 5 bp (GGATA) deletion 29 bp before the exon deletion and a 2 bp deletion (GT) 53 bp after he 278 bp deletion
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37711 | GCA AAA CTA TTT GAA AGA AGA CAG C | Common | A | |||
37712 | TGT CAT AAG GAG TTA GTA GAA GTC TCA | Mutant Reverse | A | |||
37713 | TGT CTG CAT TTG TGT TTC CTG | Wild type Reverse | A | |||
37714 | Fluorophore-1 | TGC CAC CAT TGA AGT TAA AAA TTA C | Quencher-1 | MUT Probe | ||
37715 | Fluorophore-2 | TGA TCT TGA AGA TAC GTT GAC CC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37711 | 0.40 uM |
37712 | 0.40 uM |
37713 | 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 |