Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:31482590-31482689 100bp TAACACACAAGTCCCAAGTCC TCTACTCTGCGGGCCTCACT
Mut= 124 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
AACTGTTTTATGCTCCATCATATCTAAAAAAAGTGACTTCTAAGGCATTTTATTTACTTATTGGTTTTTTGAGACAGGATCCTTTTGCTAGGTAAACCAGGTAGGCTAGGTACTGTGTAGGTCCGGCTAACCTCAAACTCAAAGTGATATTCCTGCCTCCCTCTTGAACTCTGTGATTACGGTATGTACTCCACACCTGACATGGTTTTTTAAAGAGCAACCATTCATAGTAAGGGTATTTTGTATGCCTTCTTTCACCACTCCCACTggtctgtgttttaattcctaatgtcatcagattacattttagttgaccgggtttatacagcctgctttattacttttgtaggtattaaaaccaggcagaagtgtcttattccgtgactacgggctgaatgatcacgccatgcttagatttaaagctggaagcaaacttggagaaaatttttatgtcaggcaagatggaaccagatcgtatttttttactgatggtaagaggaatggagtgtctccttagagtgtgtctttctctgctctgtgtacttgactgcaccattagtggagtgctgcctgggcaggggtgtggccgtgctaacacacaagtcccaagtccttagagcaagttctgatacaccacatacccagccctggcctgtggGGACATGACTAGTGAGTGAGGCCCGCAGAGTAGACAGTCCTCAGATCTGTCATGTGATGCCAACAGGAACTGTGCTGGAAACGGGGAAGTAGGACCAGAAAGTCACATCTTCCAAACAAGTGCCTTGAGTTCACCATGGCGGCCTGGGGACACTTTTCCAGACCCACAGGCAAAACTCCTTGACTAAATTTGCTGGCACAGAGCACTGGGAATGTGGCTCAGGTGGTGTAGTGCTGACCTGGCATTGCTCAGTTTCTGGATCCAGTCCC
This mutation is a 390 bp deletion beginning at Chromosome 14 negative strand position 31,483,013 bp and ending after 31,482,624 bp (GRCm38/mm10). In addition there is a 9 bp insertion at the 390 bp deletion site (AGTGAGTGA) and 4 bp deletion (TAGT) 37 bp before the 390 bp deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37965 | TAA CAC ACA AGT CCC AAG TCC | Wild type Forward | A | |||
37966 | TCT ACT CTG CGG GCC TCA CT | Common | A | |||
37967 | ATG TAC TCC ACA CCT GAC ATG G | Mutant Forward | A | |||
37968 | Fluorophore-1 | TAG AGC AAG TTC TGA TAC ACC ACA TAC | Quencher-1 | WT Probe | ||
37969 | Fluorophore-2 | AAG AGC AAC CAT TCA AAG GGT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37965 | 0.40 uM |
37966 | 0.40 uM |
37967 | 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 |