Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:38387438+38387548 111bp GGGTAGAACCACACATGAGC CCATTCTCTAGGAAGGAGAGTTC
Mut = 103 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AATACTACATACAAGAAACAAAGTGTAAATTCTGTGTCCAAGAGACCTGTCTGGGTAGAACCACACATGAGCGGACCCTCTtctggtagataacggctgagaggatgtaggagccccatagtgtgagctactctgagagagaactctccttcctagagaatggacgcttgctttactgtcgagcacgaccatcaccaaatgtctgtttcaggaatatgaaaatgcaacgaaggaggaaaattgcaatccagaagtgtgggtgaacttggcctgcacctacttttttcttgggatgtataaacaagcagaagctgctgggtttaaaggtaaagtgagctttttatagatcacttttgacttaatggcctgtccattgCTGTAATCCCTACTGTCTTATGGTTAGTCTTTGCTTTGTACCTAGTTTTATCTTTGCTCCTAATGTGTGTGTCTTATAGATAATTCAATTTACAGTTGAAACT
This mutation is a 295 bp deletion beginning at Chromosome 6 position 38,387,467 bp and ending after 38,387,761 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50104 | GGG TAG AAC CAC ACA TGA GC | Common | A | |||
50105 | CCA TTC TCT AGG AAG GAG AGT TC | Wild type Reverse | A | |||
50106 | AGA CAC ACA CAT TAG GAG CAA AGA | Mutant Reverse | A | |||
50107 | Fluorophore-1 | CGG CTG AGA GGA TGT AGG AGC | Quencher-1 | WT Probe | ||
50108 | Fluorophore-2 | CCT CTC TGT AAT CCC TAC TGT CTT ATG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50104 | 0.40 uM |
50105 | 0.40 uM |
50106 | 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 |