Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:76268688-76268785 98bp TGAACAATAGCCCACCACAG GCGTTCTTAGTTGACATTTTGACTG
Mut = 94 bp
Wt = 98 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TATTTAAAATAAAAACATTTATTTAAAGACAAGATGTTTGAACAATAGCCCACCACAGCATCACagggtacattttgaatctgtcatctctgttttaggtaagattatctccagtcaaaatgtcaactaagaacgctacagatctagttgagtatgttgacaagtaagtctgtaagttaagtctatttcctgcatttaatgaatcagtggagattagaatggctgaaggaaacttttcaaatcatgtaagtagctctctgctctcagaaaacctgaacaaccttagacagacaaagttcactgtttctaaacacatggtgctaccttctctttaagtgacttcataaactcctacagatctgttggggaaataatacaacTGGGGCCAGAAGTGGCACATAACACAACAGATAAACAACTATAACTTCAAATTAAGCCAAGCAGATCCACCGACAC
This mutation is a 316 bp deletion beginning at Chromosome 13 position 76,268,444 bp and ending after 76,268,759 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51994 | TGA ACA ATA GCC CAC CAC AG | Common | A | |||
51995 | GCG TTC TTA GTT GAC ATT TTG ACT G | Wild type Reverse | A | |||
51996 | GGA TCT GCT TGG CTT AAT TTG | Mutant Reverse | A | |||
51997 | Fluorophore-1 | AGG GTA CAT TTT GAA TCT GTC ATC TC | Quencher-1 | WT Probe | ||
51998 | Fluorophore-2 | CCA GAA GTG GCA CAT AAC ACA A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51994 | 0.40 uM |
51995 | 0.40 uM |
51996 | 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 |