Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:68031913+68032035 123bp GAAGGGAGACAAAGCCAGAA AATCCAGAGAGCCTGGTGCT
Mut = 150 bp
Wt = 123 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TCACAGGAGGGCTACCATCTCTGGAATTCAGCTCTGCCGTTGCTGTGGCAAGGGGGATGAACTGACAGCAGATCCATAATGGTGTAAACTGGTTCATGTGGGTGGAGGTGACCATGctaagaagggagacaaagccagaaagtgacaggccacggaaagtccggtttcgaattgcctcatctcacagtgggagagttctgaaagaggtctatgaagatggacaagcaccaggctctctggattctgaatgtgctagtatttgtgccatagatgggctaagtgactctgagggacagcaaaacggccacattggatcagaggataatgaacaggagaaagatcaggataacctgctggtattagccaggacagccagcgagaaggcttttggcacaagaagagtcaacattttaagcaaaaatggtacagtcagaggcgtcaagtacaaagtgagtgctggccaggctctgtttaacaatttgaccaaagtgttgCAGGTAAGTCATTATACACTTTCCTGTTCTGCGCTCTTCTCCTCACCCACCATCTCCACCCCTTTCAGTTCTCCAGGATGGAGTCAACGTCTCCTTCCTCCA
This mutation is a 378 bp deletion beginning at Chromosome 5 position 68,031,909 bp and ending after 68,032,286 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49112 | CAC AGG AGG GCT ACC ATC TC | Mutant Forward | A | |||
51437 | GAA GGG AGA CAA AGC CAG AA | Wild type Forward | A | |||
51438 | AAT CCA GAG AGC CTG GTG CT | Wild type Reverse | A | |||
51439 | AGC GCA GAA CAG GAA AGT GT | Mutant Reverse | A | |||
51440 | Fluorophore-1 | TCC GGT TTC GAA TTG CCT C | Quencher-1 | WT Probe | ||
51441 | Fluorophore-2 | CAG CTC TGC CGT TGC TGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49112 | 0.40 uM |
51437 | 0.40 uM |
51438 | 0.40 uM |
51439 | 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 |