Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:45722784-45722870 87bp ACCTCTTCTTTTCTCTGTCC TGAGAACACACTTCCCCT
Mutant= 99 bp
Wild Type = 87 bp
Wt Sequence (deletion in lower case):
ATCCCACCTATTTGTTGGGCATCGTAGGGCTTGGCTTCTAAGAGTCTCTGAGAAGACCCAATTCAGTTCTTTGACGCATCACAAACTTAGATAGGATGAGTTAGTGAAAACAGCAGATCCCAGAGAGCAGATCAGAATCCTCCCGGTTCCCCGTGAGCCTCCAATGCATCCTCCAttaagaatgggagatcatgtcctcatcatccaggattttcaaggagggaactaggttctggctgcaagattgtggtcagctattgtctgtgaatgggctccctgggggtcttctgtggtgtgggtgtgtgtctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtagatgcaccactcacttctgacctgtgacatttattgcaaagggaaacacactaaccctctgtcccccacactccctttttaggtcctgtgcaagctgataaacagtttatatccaccagggcaagaacccatccccaagatctcagagtcaaagatggcttttaagcagatggagcaaatctctcagttcctgaaagctgccgaggtctatggtgtcaggaccactgacatttttcaaacagtggatctctgggaaggtaaactgccctcctggctttaggtctgtctcccctggactcttgtcccagtacctcttcttttctctgtcctctatttgcaaaagccacttacacagggattccctgtgctgcTTGGGGGAGGGGAAGTGTGTTCTCAGCTGTCATCCCAGGGACAGTGCTGGCCTTCCTGTTTGTGATGCTTTCTGGGTAGGGGTCCTGTCGTACAGAAGGACACATATTGAGGCTAGGGCAAGAACCTCAGCCAAGTAGTGGCTAAGTGTGATAGTGGAAGTGTCCACAGAGAAGAAGGGAACTGGCTACA
This mutation is a 545 bp deletion beginning at Chromosome 16 position 45,722,809 bp and ending after 45,723,353 bp (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
70858 | ACC TCT TCT TTT CTC TGT CC | Wild type Forward | A | |||
70859 | TGA GAA CAC ACT TCC CCT | Common | A | |||
70860 | Fluorophore-1 | TTT GCA AAA GCC ACT TAC AC | Quencher-1 | WT Probe | ||
70861 | TAG TGA AAA CAG CAG ATC CC | Mutant Forward | A | |||
70862 | Fluorophore-2 | AGC CTC CAA TGC ATC CTC CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
70858 | 0.40 uM |
70859 | 0.40 uM |
70861 | 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 |