Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:31764235-31764327 93bp TGAGAAGAGAACCCCTTTGC AGCTCAGGAGTTGCCATACC
Mut= 107 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TAACTGTACATAATATCTTTAAGAATTTTACATGTGAACAAAATTTCTTCAAAGTTAGTTTTTTATTTATGGTTACACATCACTTCTCGAAACCTTTTGGGATTTGTTTCATTTCAGATATTTTAaaaataggactgggtggtttggctcaagcCATGGTTTTATAGCCAAGTAAATTTGTAAAGGCTTTTACTTGTCTTAGCTGTAAAACACCCAAGattttgttggcgagacttgcagagtagagttgtggaaatattaatttgttagttaataatgttggatttcattcatactacattattttttattaaagtgcagtttcctcttttcacaggacaatgagaagagaacccctttgcatgctgctgcctatcttggtgacgcagaaatcattgagctgcttattttatctggtatggcaactcctgagctgagttcatggtgttcagtttgatgtatgagtgttcctatggaatgcatagcctatgtcttagtattggtttcataagttaaccaattataatagcaaactttgatcatagaacctgttttgggtgcttaaagtactttaagaaatagaacaaagatttctgtccttacAGACTTATTAGTTCCATAAATACAAGTCATTTTATAGTTTGTTTAAAAGATAATAATTGCTACTGTGGAAAAATGAAAAACATAGATTGGGAAGAATAGGAAAGAGACATATGTCAGGAAAGG
This mutation is a 385 bp deletion beginning at Chromosome 14 position 31,764,067 bp and ending after 31,764,451 bp (GRCm38/mm10). In addition there is a 29 bp intronic deletion, Chr 14 :31,764,516 -31,764,544, which is 64 bp before the 385 bp deletion, that will not alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39518 | TGA GAA GAG AAC CCC TTT GC | Wild type Forward | A | |||
39519 | AGC TCA GGA GTT GCC ATA CC | Wild type Reverse | A | |||
39520 | CGA AAC CTT TTG GGA TTT GT | Mutant Forward | A | |||
39521 | AGT CTC TTG GGT GTT TTA CAG C | Mutant Reverse | A | |||
39522 | Fluorophore-1 | TGC TGC TGC CTA TCT TGG T | Quencher-1 | WT Probe | ||
39523 | Fluorophore-2 | CAG ATA TTT TAC ATG GTT TTA TAG CCA AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39518 | 0.40 uM |
39519 | 0.40 uM |
39520 | 0.40 uM |
39521 | 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 |