Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:72941187+72941310 124bp TCTTGCGTGGTGAGTAGTTATGA CTTTGGTGATGAGTACAACCTAGA
Mut= 118 bp
Wt= 124 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTTGTAGCTCTAATCACGGAGAAAAGTAGAATTGATTTTGAGAAGTCCTCCTTTGGGTTCATTTGTGGTTGAGATTTGGGGGTTACGCTGCTTGCAGTACCATTTTCTTGCGTGGTGAGTAGTTATGACTAAGCGCACCCCATCATTCACCCCTActttatagtcctatactcagtaacaaatgtgccctttggtttcattttcttctaggttgtactcatcaccaaagaaaaaactcacaccaatgcagaaatctgtcagtccattagtttggtgcaggcaagttttggattacccaagtcctgatgtggagtgtgctaaaaagtcccttatccacaaacttgatcaaactatgtcaggtatgtctataacttaaattgtgaattcattaattttctttggcctgaaagcatatatgttgagtgtgtatatttaggcaggcAGTTCTTGCTTTATAGCCTTTCTGAATTTCACTGTATTCATCATGAAGGCTAGATTCATGTGATGACAGTTTTTAAAATCTTAGTCTTACTTTAGTGGATTATATCTATGAGATTATGAATTTTAATTTTTCTAATAATGACCTAAAACAGTTCCTAAAATGAAAATGTTTGTC
This mutation is a 287 bp deletion beginning at Chromosome 5 position 72,941,237 bp and ending after 72,941,523 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44103 | TCT TGC GTG GTG AGT AGT TAT GA | Common | A | |||
44104 | CTT TGG TGA TGA GTA CAA CCT AGA | Wild type Reverse | A | |||
44105 | CTG TCA TCA CAT GAA TCT AGC C | Mutant Reverse | A | |||
44106 | Fluorophore-1 | CTC AGT AAC AAA TGT GCC CTT TG | Quencher-1 | WT Probe | ||
44107 | Fluorophore-2 | CAC CCC TAA GTT CTT GCT TTA TAG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44103 | 0.40 uM |
44104 | 0.40 uM |
44105 | 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 |