Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:23885671-23885777 107bp TGGCTGAGACTTTTACTGGAAAG TTTGCCAATCCTGTTGAAGA
Mut= 100 bp
Wt= 107 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGAAGAAGAAGAAGAAATATCTAGTAAGCACTTGGAATTGGCCCTGGAAAGAGGTAGAAGTAACTTTTTAATTATGATACAATTAAAGAAATAAAAGTGAAATCTTTCCATTTGTTCTTATAAAATGCTCCCATCAAGAATGACCTCACTCTGAAGAAGGCTAAAAAGCATGTAACAAAAACtaaagagaagacggggattggctgagacttttactggaaagagctgcagttcattaatttgatagttgagttccttaaaggggccttaaagtgcttttacacatctcttcaacaggattggcaaatgtacagtgtatcagttgtgcagtaaaagactggaaacttgtacatgttctttatgcaaagtaacttaaatgattgaatgccttttgctttattttagaatctcccaccttctgttgtggctactgttggtggtaaaattttcacatttggatcttacaggcttggagtacacaccaaaggtaattgctttttagttatgttctagtgataagaatttttaggaaactgttttacttgtttacaattagcgagtactgaacatagttcccatatcttactacTGTTAATAAACACTGTCTTATACTCATCTTTCATACCTAGTTCTGCATTTATGTTTATGTAAACTTTAATGAAAATATAATTTATCATTCTTAAGAAATAATTTTATTTTTTTAGGAGCTGATATTGATGCACTTTGTGTTGCCCCAAGACATGTAGAGAGATCAGATTTTTTTCAGTCCTTTTTTG
This mutation is a 407 bp deletion beginning at Chromosome 11 position 23,885,389 bp and ending after 23,885,795 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47356 | TCC CAT CAA GAA TGA CCT CAC | Mutant Forward | A | Mut F | ||
47358 | GCA GAA CTA GGT ATG AAA GAT GAG T | Mutant Reverse | A | Mut R | ||
48713 | TGG CTG AGA CTT TTA CTG GAA AG | Wild type Forward | A | Wt F | ||
48714 | TTT GCC AAT CCT GTT GAA GA | Wild type Reverse | A | Wt R | ||
48715 | Fluorophore-1 | TTG AGT TCC TTA AAG GGG CC | Quencher-1 | WT Probe | ||
48716 | Fluorophore-2 | CTG AAG AAG GCT AAA AAG CAT GTA AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47356 | 0.40 uM |
47358 | 0.40 uM |
48713 | 0.40 uM |
48714 | 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 |