Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:46832399-46832571 173bp AATATAGGGGCATCGGGTAA TCTTGGCGTCTTCCAATATC
Mut= 164 bp
Wt= 173 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case:
TGCTCTTATGATAATCAAACTCATTGAACTAAATGTGCCCCAGCATTCAATAAAACATTTCTTCTGCAATGCCTTTGGCAGGAAAAAATATATGTGCTAATGGATTTTGAGTCACTGATACTGCAGTTTGGtaaggcagagcccttctgatcctgtatgaatcttgggaacatggttgagtcatatttcatgtgttggaataggaaaaagaactctagaaattctctagtaatctagtagtatatctccctattttaattttttgcataaattcctatggaaagcatggctcttatgtgtttgtattacagtatttcaagtgatatttagagtattttaattgtacaataatattttctgcataatcttaaaaacctgcttatactcagtttgttttatccgtttccttgcagtgtttgaacgtcactcagttgttaaaacactttggtcttggtccgaactctcccatctcaccggatctattcacgtacctttgccctgcattgctgtatcagatcgacagcagactttgtattgaacattttgacaaacttttagttgaagatttaaataaggataaaactctggttcctgaagataagacaaatataggggcatcgggtaagaaagacttttttcctctttaaggtagTACTCATACTGTCCCTTAAATTATTTATTAAAAAGAAAGACTAAATTGTTAGTTTCATTTCAGTGTCAGATTTTAATGATCCAATCAGATTTAGATATCTATATAAGATATTGGAAGACGCCAAGAGGCTTATTAGTATTTTTATTTTATTTCCTCTGGCCCTG^TAGAGGAGTGTTTTGTTGAAAACAGAGAAAAAGATTAAAAAGGAAATAGAAATCTTTTTGACAATCAGCCATAAAATGGGAACTCTGATTAAAGTT
This mutation is a 521 bp deletion beginning at Chromosome 1 position 46,832,525 bp and ending after 46,833,045 bp (GRCm38/mm10). In addition, there is a single bp (A) insertion 164 bp after the exon deletion that will not alter the results of the exon deletion
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40956 | AAT ATA GGG GCA TCG GGT AA | Wild type Forward | A | |||
40957 | TCT TGG CGT CTT CCA ATA TC | Common | A | |||
40958 | GTG CTA ATG GAT TTT GAG TCA CTG | Mutant Forward | A | |||
40959 | Fluorophore-1 | TAA GAA AGA CTT TTT TCC TCT TTA AGG TAG | Quencher-1 | WT Probe | ||
40960 | Fluorophore-2 | TGC AGT TTG GTA CTC ATA CTG TCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40956 | 0.40 uM |
40957 | 0.40 uM |
40958 | 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 |