Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:44708500+44708641 142bp AAAGGTTCAGGAAACTAGACTG TCAGGCGTATGCATCCATGT
Mut= 140 bp
Wt= 142 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCTTTTAGATTATGCAAACCCTGTTGCCAAAGTTATCATAACTATAGAAGAACAAAATATTTTCTTATCTGTCAATTATTAAATATATCTTGGCATCTATAAAAGCAGTAAACACAAAATAAAAGTTGTCTTAATTTGCGTAACTGTTATTACTCATAGCTCCCTGCTGATCTTATGAGgtgaggaccaggcgaagtgatatatgtgtaaaagtacagcaagtttggccttagccttggctccatactggtattgcacaaaattaacagatgtattttgtggttaaaaaaaaaagaataaatgccttgatcatttccagttacaatatttctttctttaactaacaaaggttcaggaaactagactgtgctccccagcaagacttccttcaggtgagaatgtatgtatatgtcatcatagaaatttgtggtttaattcagtactgcattttctctgttacagacaaaacatggatgcatacgcctgaagctttatcaaaacattacattccctataatgcaaaggtaaaatggttattgagatattatctatttttacattgttatatgttatgttgaaataattgtgctaattaaaaaaaaaacttatgcattggcatgtcctgGTAAGCAATATTTGAAACTAATTTCATATTACTGAAATAAGTAATATAACATAAAAAATTGTTTTTATATCCAAATTTCTTTGAAAAGAAAGGGATACTCTGTTTAGATTCTCTATAGTCATGTATTTCACTGAAGCTTTAAAAAAAA
This mutation is a 446 bp deletion beginning at Chromosome 1 position 44,708,334 bp and ending after 44,708,779 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45638 | AAA GGT TCA GGA AAC TAG ACT G | Wild type Forward | A | |||
45639 | TCA GGC GTA TGC ATC CAT GT | Wild type Reverse | A | |||
45640 | TCA TAG CTC CCT GCT GAT CT | Mutant Forward | A | |||
45641 | AGA GAA TCT AAA CAG AGT ATC CCT TTC | Mutant Reverse | A | |||
45642 | Fluorophore-1 | CAA GAC TTC CTT CAG GTG AGA AT | Quencher-1 | WT Probe | ||
45643 | Fluorophore-2 | ATG AGG TAA GCA ATA TTT GAA ACT AAT TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45638 | 0.40 uM |
45639 | 0.40 uM |
45640 | 0.40 uM |
45641 | 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 |