Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:94303412-94303531 120bp AGTAACTAGCTCCAACCCAGTG GGAGTCTCTTCTACCCATGCTC
Mut= 131 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCAGCTATTTGAGAAACTGAGACAGTAAGATCTACGTTCAAGGCCAGTCTGTGCACTTAACGGGGAGTTTTATCACAAAACAAGGGGCTGGAAAATACAGCTCAGTGCTAAAGCACTTTGCTAACTTGTGTTCAATATATAGTACCACCAAAATAAACAGCCAGCTGATTATTAACCAACTGCAATACAGTATATAGTGCCTTTCCCtttgataggataacatctaaaaatatacttcatgtaagtttaggctttgagacggggtgatagggaatatatatatatggcttgtaatgtttaaaagtttatttgttttcactttatcaggtatgagaagagttctcgctttatgaaagttggctatgagagagattttttgcgatacttacagagtttacttgccgaggtagagcgcagaattagacgaggtcatgctcgtttggcattatctcaaaatcagcagtcttctggggtgagtatgaaatggacttagagtttgtaaagaactctgttctctgtagatgtggctgtctcatatcattttaaactcgacttccattcattactctgtcctttagaatacataaggtttgaggagcaccttccttctgttgtttgagggttagggaagcgtctccccccttccgcgtgcatgtttggtctgatggtgctgctggaggtcatgggctttgtatatgctagcacgtgctccaccactaaaccacacgttcgttctccagcctctgctccatgctaggaagaccagggcgggtggtcatcctagtaactagctccaacccagtgacttacttcacctccaccatgagaataggataagcaaaggggccaagactgtgtaaggagatTGCTTTTCTGAGGAGAGCATGGGTAGAAGAGACTCCTTTCTGTGACCTTTGAAGGCAAGCAAGGAGCTAGCTAGCCAGCAGAATCAAACGGAAGCCGCTCGGGTTGGGCTGCAGTAAAGCATAAAGACAGAAAAGGAGAGGAGGTTATTTCCCCAGACAGCTGTGACAAAGCTAGAGTGCAGGTGTAAAGATCTTAAGTATATTTCT
This mutation is a 659 bp deletion beginning at Chromosome 11 position 94,303,448 bp and ending after 94,304,106 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47683 | AGT AAC TAG CTC CAA CCC AGT G | Wild type Forward | A | |||
47684 | GGA GTC TCT TCT ACC CAT GCT C | Common | A | |||
47685 | GCA CTT TGC TAA CTT GTG TTC A | Mutant Forward | A | |||
47686 | Fluorophore-1 | AAG CAA AGG GGC CAA GAC | Quencher-1 | WT Probe | ||
47687 | Fluorophore-2 | ACA GTA TAT AGT GCC TTT CCC TGC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47683 | 0.40 uM |
47684 | 0.40 uM |
47685 | 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 |