Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:83703711+83703838 128bp ATGTCCAGGAAGCCACCAGT CTTGGCCCTTGTGTGAATGA
Mut= 128 bp
Wt= 128 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CATCTGCAGGTAGGAGGGGCCCAGATCCACAGTGTTTCAGATTCTCATCTGTAGAGCTGTAAAGACACAAACCCCATCTTTCCCAGAAGAATGCTACATCATTATGTAACTGGATGTCCAGGAAGCCACCAGTGGGCCCTGACACTCCTTACTGGCTGTCTCTGCTCCAGctcttactgtgagacacagtgctacacagcaataaatatttcccttgctcatcattcacacaagggccaagatgtggtgtaacaactcacatggtaaggaaatgagtttgagactattttgtgattttgtttagcatctatcaaagaccaattagagagaagtcacttatgaaactggaaaaaggaacctggtgatgggcaggcactttctggaaagctcttgttttcatgcatttccccattgactgcttcctcccacacccataattctagctttactgaggctataaattatttcatgagttcagccgagccagagccaacatgaagacagccacagtcttgtttctggtggctttgatcactgtggggatgaacactacctatgtagtgtcttgccccaaaggtaagtgtgttaagggcatccaagctggaaagagaggggactctagaagatcactaggaccaggggatgcattattacagggaaagCCATGTGACAACTGCTTGGATCACACTCACTCCCTTTAGTCTATATAGAGTGATCATTCTGCATTCCTTAGCGTTCTTAAAGACAGAGGCTGGCAGGAGGGGGTTTAGTTTGAGAGAAACTTGCACAGAAATCAGGACAGCTCCGCTAGTTTAGAGATGTGCCTTGCATTAGAGTCCTGTGGGAGCACTTGCTCAGAGATGAA
This mutation is a 492 bp deletion beginning at Chromosome 11 position 83,703,768 bp and ending after 83,704,259 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47559 | ATG TCC AGG AAG CCA CCA GT | Common | A | |||
47560 | CTT GGC CCT TGT GTG AAT GA | Wild type Reverse | A | |||
47561 | CTA AGG AAT GCA GAA TGA TC | Mutant Reverse | A | |||
47562 | Fluorophore-1 | ACA GCA ATA AAT ATT TCC CTT GCT C | Quencher-1 | WT Probe | ||
47563 | Fluorophore-2 | TGC TCC AGC CAT GTG ACA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47559 | 0.40 uM |
47560 | 0.40 uM |
47561 | 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 |