Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:35114398-35114496 99bp CTTGTCCATTGCCGACCTT CTAGGAAGGGGAACACATCAG
Mut= 70 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
AGAGAGTGGCATCTCTGAAGGGGAGGCGGGGTATGCAGACAAATGAATTGATTTATTTTTTTTCTGCCCCCCCCTTTGTGTTTTTGTTTTTGTTTTTTTAATTTGTCTTTGAGAATTCATGGACTAAAGTCAGGTCTGCGTGAATGCCAGGAAGATAGAGGTTCGGGAACAGTGGGAAGCTATTAACCAGGGTTATAACTGTCCCCTCTCCTCTAGATACTACTGTTCGCTTCcacgtacggggctgtggacagcatcattcctaccgatgtcaagaaaggcatgtgatctaccaatcagactacttgtataaggctgattgctgccagtacgattactgcaactcctggtccagtgctcagcaccagagcaccctgcgtgggtccccaggaagccatctgggcatgcccctgtcagcgtctcagataaaacagttttaccaggccctgaacctctctctgcctcagcctggcttccatgctcataaggtgtctgagggcctggagtctctcattctgcccccggagctgggcttgtccattgccgaccttcgccaaatatacttgttcctcaacagttcaggacttctggtccttccCTGGGATAGACCCTGATGTGTTCCCCTTCCTAGATTCTGCTCTGTCTGAGAGACTCTTCCTTTGAGGGGCCCAGCATCCTGCTCTGCGGTCTGTGAACACTCAGATCCTCTGAGCCCTCTGATCTCTTAAGCTTTCTGCTCTACATGGCCAAATTCTGTATGGCCCCTGGATCTGGGGCTGGTTTGTCCTCCTCCTTTTCAGGAAACTTAAGGACGGTTCTCACCAAGGCTGACTTGACAAT
This mutation is a 365 bp deletion beginning at Chromosome 17 position 35,114,431 bp and ending after 35,114,795 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64375 | CTT GTC CAT TGC CGA CCT T | Wild type Forward | A | |||
64376 | CTA GGA AGG GGA ACA CAT CAG | Common | A | |||
64377 | AAC TGT CCC CTC TCC TCT AGA TAC | Mutant Forward | A | |||
64378 | Fluorophore-1 | CAG TTC AGG ACT TCT GGT CCT TC | Quencher-1 | WT Probe | ||
64379 | Fluorophore-2 | ACT GTT CGC TTC CTG GGA TAG AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64375 | 0.40 uM |
64376 | 0.40 uM |
64377 | 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 |