Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:21781122-21781223 102bp GGGACCTTGAGTGTTCTCAG AACCCAAAACAAACGAAACC
Mut= 97 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ATCCACGACTGGCGCCTAACTTACATGACTGCACAATGACTTACATTCTTTTTCTTGTTGGTTGGTTGGTTGGTTTTGGTTTTGTTTTGTTCTTTTGATATGGGGTTTCTTGGTGTAGCCCTAGCTGTCCTGGAACTTGGATCTGTAGACAAGACTAGCCTTGAACTCATAGAAATATTTGGGCCTCTGCCTCCCAAGTGCTGGGACTTAAGGCATGCTGCATCACCACTCGCCTACTCATTTACACAGAGCCATTCTGTACAGAAGCTCTGGATTCTGCTACGGATGCTGCACTTCCGTTTGCTTGCTTGCTTGTGCTGCTAGAGATGGAACTCGGGCTCATTTCATGCTAAGCAGGTGCTctgtggctccagcctgaagcctcgctttggaaaaattttaatgatattaaaacatgagggttggatgtactctgttcagatttaactcccttttgtgtgtttatcagaatcaagatcattatgcggttcttggacttggccacgtcagatacacagctacacagagacagatcaaagcagctcgtaagtacttacttagtatgggaggtataactccaaactcgggggtgcagcaccctgaagttcatgagaccccatcttagaaagggaagggattagggggaccttgagtgttctcagagttcaagtcctgcaggaagagatcagaaaccctgtaagaatCATTGCCCATCAAGGAAACGGGTTTCGTTTGTTTTGGGTTTGGATGGTGCTGGGAATCAAACCTGAGGCCTTGAGTCCTGGCCAGCTCTGCCTGCCACTGAGCTTCATGCCTAGTCCTCTGCTCTGAGGATTTCTTCCAGGTAAGAGAGAAACAGGGTCTTGCTGTTCAGACCTTAGATCCATGTCCGCCTTCCAAGTGCTGGGATTGTAGACGGTTAGGTATGATCACCATGTTCTCGTCTAATTAAAAAATTAGGTCTCTTTTAGTTTTGCTTCCTAAAAAAGATTTTAATTTTGAAAAATTAGATGTGTGTATCTGTGTGGGTATGTGCACGTGAGTGTAGGTGTGTATCTGTGTGGGT
This mutation is a 3625 bp deletion beginning at Chromosome 19 position 41,924,589 bp and ending after 41,928,213 bp (GRCm38a 342 bp deletion beginning at Chromosome 5 position 21,781,162 bp and ending after 21,781,503 bp (GRCm38/mm10). mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48298 | GGG ACC TTG AGT GTT CTC AG | Wild type Forward | A | |||
48299 | AAC CCA AAA CAA ACG AAA CC | Common | A | |||
48300 | TGC TTG CTT GTG CTG CTA GA | Mutant Forward | A | |||
48301 | Fluorophore-1 | AGT CCT GCA GGA AGA GAT CAG A | Quencher-1 | WT Probe | ||
48302 | Fluorophore-2 | CTA AGC AGG TGC TCA TTG CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48298 | 0.40 uM |
48299 | 0.40 uM |
48300 | 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 |