Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:117702466+117702563 98bp CTTTGCAATGGTGTGACTTTTG GGCTCTATTCTGGCCTTTGT
Mut= 105 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGGCCAGCCTGCTCTGCACAGTGAATTCCAGGACAGTCAAGGTTCCATGTCTCAAAGAAACTAAGGAAAAAAAAAAAAAAAAAACCCTAAAATACTTAGAAGTTTGCTTATAAACCTTCATCAGTGTGGACATTGCTACAATTGTTTCTAAAGAAGTTCTGATTTTTTTTTTCAGGCTGCTGATCAAAAAACcaaaggtaagtatgtttgtttgtttatttttatactttgtgaggggcatactttttatagtagtatcaaactttgcaatggtgtgacttttgtaaaatttatgtttataggtgttttgcctacatgtttgtctgtgtacCAAGTGTGTACAAAGGCCAGAATAGAGCCTTGGATCTCCTAGACCTGGAATTGAGATAGCTGTGAGCCACCATGTGTGTGCTATTAAATACTTCTTTTTGTCTTCCCTTGGACACAATTGCACATGTAGATACTTGCATTGTAAGGACTTTTGCAAAG
This mutation is a 139 bp deletion beginning at Chromosome 11 position 117,702,396 bp and ending after 117,702,534 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42148 | CTT TGC AAT GGT GTG ACT TTT G | Wild type Forward | A | |||
42149 | GGC TCT ATT CTG GCC TTT GT | Common | A | |||
42150 | TTC ATC AGT GTG GAC ATT GCT AC | Mutant Forward | A | |||
42151 | Fluorophore-1 | AGG TGT TTT GCC TAC ATG TTT GT | Quencher-1 | WT Probe | ||
42152 | Fluorophore-2 | CTG ATC AAA AAA CCA AGT GTG TAC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42148 | 0.40 uM |
42149 | 0.40 uM |
42150 | 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 |