Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:100076198+100076342 145bp TGGACATCGACTATGAGGTGA TCTTGACCTTGGTGCAGTTCT
Mut= 143 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCTTTGGCTAATACTGTAAGTATTTCTGTTACCAGGTGATTGATGGTGTCCTGAAAACATTACATTTCCTGTGTGGAAGTGGGGGTGTATATCATTTGAGCTAGAACCTGGTTCTGGAAACTGAGACGAAAGCCTGTTGCATGACATTTTCCAAATGCTCAGTGGTATgccctgtagagggacagcttgaactgagttagtttgagggaacatcaggttcctggtactcagtgctgcagataattaggattcaaagttgacaggctaagactctattccatcttcccctatgcagatgatagcaaagcccgcctcagtagtattacaccctcagccattctgggtagccttctggatgaccagcactggcactcagttctcctggagcgtgtgggaaagcaagcaaacttcactgtggacaggaacacccagcactttcggaccaacggtgacactgatgccctggacatcgactatgaggtgagacaatcctcccaatatgttccaaggagtgggagggaggttgctatataaaaattttaagccactttatCTTAGCTGGGCTACCAGGGAGAAATCTCTGATGAAGAACTGCACCAAGGTCAAGAAATGTCTGGGCCAAACTCTGTGTTCCTGATGAAGTGTCAATTGTACAAGCTAAAAACCAATACTTCTATTTACTTTCTCTGTGTGTAACTATGTATGTATGTATGTATGTATGTATGTAT
This mutation is a 385 bp deletion beginning at Chromosome 1 position 100,075,903 bp and ending after 100,076,287 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46239 | TGG ACA TCG ACT ATG AGG TGA | Wild type Forward | A | |||
46240 | TCT TGA CCT TGG TGC AGT TCT | Common | A | |||
46241 | GGG GGT GTA TAT CAT TTG AGC | Mutant Forward | A | |||
46242 | Fluorophore-1 | CCT CCC AAT ATG TTC CAA GGA | Quencher-1 | WT Probe | ||
46243 | Fluorophore-2 | CTC AGT GGT ATC TTA GCT GGG CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46239 | 0.40 uM |
46240 | 0.40 uM |
46241 | 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 |