Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:88125198+88125312 115bp ACTGGATGTAGTGATGTTAATTATGAG AGAGCTTCCCTTAAGCAAATGA
Mut= 112 bp
Wt= 115 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCAGGAAGTTGCTGTTGTCCTTCAGTTCCCAGGGTTCAGAATGTTGAAGAGAGTCCCTTAAATGGCAAGTCTGACTCTTGATCCGCTTCCATCAAATGTTGTGGTCTTTGGCTGTGTCTCTTAAGCACTGCTGGAAGTGGTATCAAGActgaaggggtggagggtgtgtctgcttactaattttgtgtttatctctcatcagtgcttcttatagtggtgtctgtctgtacagctactggtgcctggaactggctaatagatcctgagacacaaaaggtagaaattttgttttaaagtctttagcagattaaataagatactggatgtagtgatgttaattatgagatatgccgagctacctttacctgctaCTAATAATGTTAATCCTTATATTAATAACATGCCCTGTAATCATTTGCTTAAGGGAAGCTCTAGTTGGCTGTTGTGAGTTACTTATCTTCATAAGAGAAAGATCTAAGAAAATTTACTCTGTTTGATATTTCTTAACTCATGTTTTTAATTTTTCTATTTCCAC
This mutation is a 223 bp deletion beginning at Chromosome 8 position 88,125,028 bp and ending after 88,125,250 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43450 | ACT GGA TGT AGT GAT GTT AAT TAT GAG | Wild type Forward | A | |||
43451 | AGA GCT TCC CTT AAG CAA ATG A | Common | A | |||
43452 | TTG TGG TCT TTG GCT GTG TC | Mutant Forward | A | |||
43453 | Fluorophore-1 | ATG CCG AGC TAC CTT TAC CTG | Quencher-1 | WT Probe | ||
43454 | Fluorophore-2 | ACT GCT GGA AGT GGT ATC AAG ACT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43450 | 0.40 uM |
43451 | 0.40 uM |
43452 | 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 |