Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:104430159-104430251 93bp TCATTTCATGCCGCTGCT GCCTTAAGCAAACTGCTCTCAC
Mut= 90 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCCACAGCACTATTCTTAAAACCAGAACACTGAAAGCCTGTTTAGGTTGGATGAAGCTGAAGGTGTCATGTGCCCATGCAGCTCACAGCTCAGTAGACAGAAGAGCTGGTGTTTGCATCATAGTCCCTAAACCCTAAAGCTGCAACCTCtgccatgcatagatgcttttgtttatatttattattacctttttatccccttaatttgctaagcctaagatgtgtttctatttgcgtctgtataatttttttcttttccctctgtagttatgaaagagtttcaagattatatcgagcctgaagaaggttgtcagggttccccacagagaagagggcctctgacctcaggctcagacgaagacagtgttgccctccctctgggtgacaacgtgctgactcataacctggggatcccagtgttggtggtgtgcacaaaggtacgtccagggaattgtgagcttggctgggtttgcccagtgaacaagaaagagttttagaagcttcgatggagtttcttctcaagggaaaattgggcttattcaggattcatcaaggtatagtgaatgtgtcagggtaggacattctgtcatttcatgccgctgctgaattgacagtacctagttcagaaagccctgttgacTTCATGGGCAAACTCAAGTGAGAGCAGTTTGCTTAAGGCTCTAGTTTATCTGGTCCAGACTGGTGGTGACATAGAATAATGAAGTAGAAATAGAAATTAAGCTTCATTTTAGAGAAAGTATGTCTCTTATAAGAGGGGGAAGGTAGCTTTAAAAACATCCTGGATTTC
This mutation is a 490 bp deletion beginning at Chromosome 8 position 104,430,198 bp and ending after 104,430,687 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43381 | TCA TTT CAT GCC GCT GCT | Wild type Forward | A | |||
43382 | GCC TTA AGC AAA CTG CTC TCA C | Common | A | |||
43383 | AGA AGA GCT GGT GTT TGC ATC | Mutant Forward | A | |||
43384 | Fluorophore-1 | CAG TAC CTA GTT CAG AAA GCC CTG | Quencher-1 | WT Probe | ||
43385 | Fluorophore-2 | AAG CTG CAA CCT CTT CAT GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43381 | 0.40 uM |
43382 | 0.40 uM |
43383 | 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 |