Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:56010921+56011030 110bp CCTGGGCTCCAGTTTGTACT CCTCCAACAGCAGTTCTTCC
Mut= 119 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TGGGCTTTGATCCTGAAGGAGGTGGGGGTCATGAATACTATTGGCAGAGGGAACTGCTGCAGGGAGACTGTCAGGGGAGAGACAGGGCTCCGAGACAGTCAGTCTCACCCGAAGTAGTACAGCCAGGAGAAGACAGGCTGAATCCCAAGCGTGGGGAGAATCCGCACCTCGGACACCTCTGCTCACCGTCGCCctctggcggttggtccaggcaatggcacctcgggctcaagcctgggctccagtttgtactgagtgaagctcccagctgatgttgttgctacactccattcccaccagagtcccaccagtgaaggggaggaggaagaactgctgttggaggaggaggaagaagatgtgctggctggggtctcatcagaggacaaaggccacagaccccctgggaagggttccttggagccaggtgaggggccagggcagaggcactatgatgtgggaagatggctttgatgtttcttaggcctggggaatcctgaatcctgattcatattcatattctctctctctctctcattctctctctctctctctctctctctctctctctctctttctgtctgtCTCTGTCTCTGTCTCTCTCTCTCTGAGACAGGTTCTCATTGTAGCCCTGACTGGCTCAGAACTCACAGAGATGCATCCTCTGAGTGCTAGGGTTAAGGGCATGTATTATCTATTTATTGTGATGGGGCATGTAGATCAGAAGTTGGGTCCCTTCTTCCACCATGTGGGGTCTAGGAATCAAACTCAGGTCATCACACTTGGTTGCAAGATCTGCTGAGCCATCTCGCTGGCCCTG
This mutation is a 389 bp deletion beginning at Chromosome 17 position 56,010,881 bp and ending after 56,011,269 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64346 | CCT GGG CTC CAG TTT GTA CT | Wild type Forward | A | |||
64347 | CCT CCA ACA GCA GTT CTT CC | Wild type Reverse | A | |||
64348 | AAG ACA GGC TGA ATC CCA AG | Mutant Forward | A | |||
64349 | GCC AGT CAG GGC TAC AAT GA | Mutant Reverse | A | |||
64350 | Fluorophore-1 | ACC AGA GTC CCA CCA GTG AAG | Quencher-1 | WT Probe | ||
64351 | Fluorophore-2 | AGA ATC CGC ACC TCG GAC AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64346 | 0.40 uM |
64347 | 0.40 uM |
64348 | 0.40 uM |
64349 | 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 |