Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:35186591+35186725 135bp GCTTCTCCTTGCTGGTAAGTTG GCAATGCGAACTCAGGTATG
Mut= 141 bp
Wt= 135 bp
Fam=Mut
Hex=Wt
HOM Lethal (DJL 7-29-19)
Wt Sequence (deletion in lower case):
AAGGAGGTTTTAAGGGGCCTAATGAGGTGGCTTAGTACTAGAACACTTGCCTAGTGTGTGTCCATTTGTGGGTTACATCCCTACCACCACACATATAAAAAGAGTTTTGTAGGATATAAAATCATGTTACTAATTTATGATTCACTCTACaaagtaatactattgcttgttacttaatatagttcatatgtccatttgaattattgtatgtaactagaacattcacttagatatgtggaaatcagaatagtatttgtatttttcctggtttttatggagcttagcagatagttttgagaaggtagtactgggaaagatgaaggtggtgccacgtgttgcctcatatcccctagttctggataactagataagaaacacttcagtttgtcatgctaccaggcttcattgtttttgttcgcatgcatgctggccatgactctacttgcctggcctccaaatataggattaaaggcatgtgctgccttggcaaacgaggctgcgttgtcatgaactgacttctgctaggagcactattttcattacttaattataattttatcattctagcccaaaaatgttcaacagcatgagaaaattcagaaagccagttcagagggtgttgcgatccaggggcaacaaggaaccagacttcttcctgagcagctcattaaagaagcatttatcatcagtgacctttttgatattggggaattgtcagctgttgagcttctccttgctggtaagttgacatttagctggaagtgtgatatgtagtcagaatcatgaagctagaacccctaTCTTGGACTTTGGTTTTCTTTTAAAGATTTATTTATTTCGCATACCTGAGTTCGCATTGCTGTCCTCAGACACACCAGAAGAGGGCACTGGATCCCATTAGAGATGGTTGTGAGCTGGGATTTGAACTCAGGACCTCTGGAAGAACAGTTAGTGCTCTTAACCACTGAGCCATCTC
This mutation is a 605 bp deletion beginning at Chromosome 6 position 35,186,016 bp and ending after 35,186,665 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38394 | GGG TTA CAT CCC TAC CAC CA | Mutant Forward | A | |||
43456 | GCT TCT CCT TGC TGG TAA GTT G | Wild type Forward | A | |||
43457 | GCA ATG CGA ACT CAG GTA TG | Common | A | |||
43458 | Fluorophore-1 | AGC TGG AAG TGT GAT ATG TAG TCA GA | Quencher-1 | WT Probe | ||
43459 | Fluorophore-2 | ATG ATT CAC TCT ACT CTT GGA CTT TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38394 | 0.40 uM |
43456 | 0.40 uM |
43457 | 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 |