Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:59595196+59595296 101bp TGCAAGCATAATTGAAAGCA TCCAGACTAAAAGGAAACATGC
Mut= 87 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
CCGCTCGCCCCCGCCCATAGGATTTTTTGTTTGTTTGTTTCTCATTACCTACCGTTGTGAGCTACCATGTTGCTGGGAATTGAACTCATGACCTCTGGAAGAGCAGTCAGTGCACTTAACTGCTGAGCCATCTCTCCATCCCCAAAAAATGATTTTTAAATTTTTAATTCTTACTGGAATATTAAGATAACCATCTTAATTTGCTTGTTAGTTTTCAGTTCTGTGGTTTTATATTCTTTTTTCTTtctTTTTTTTTTTTTTTGATCCATTTCATAATGCAAGCATAATTGAAAGCATCactgtggtaattgagtatctagtcaatccttagaaaatgatttttaaattaatccaagcatgtttccttttagtctggaaacttaaactttattgcgctattaaagtaacttttctctctgattcttttagagctgaagttgggagaactgcttcatgataagctgtgagtatttgtggtgtagtagttgtggaaaactttaatctttttttttcaggtagaatattctttgaagtccaagtaaatttatgtttcaaatgagttgtacaatttctatttcagtttgggtgagctatcttgagtcacTGACATGGTTAAGATGTGAAACGCCAAACTGTTAGGATACTTTTCACGAAGATGGACATGCTGAACATGTGATTTTTGAAGACAGCTATTAGTTGTATGTCTAGTATGGTAGATTTATCAATGACTGATGCTGGTGTGGAATAAAATTGAATATCTCCAAGTGAACCAAAGGAAAGGAAATCCTGGCCTATATTTATACCTCGCTGTGCTGCTGCAGACACACTGTGGCCAGGC
This mutation is a 306 bp deletion beginning at Chromosome 13 position 59,595,218 bp and ending after 59,595,523 bp (GRCm38/mm10). In addition, there is a 3 bp intronic deletion (TCT) 41 bp before the larger deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48068 | TGC AAG CAT AAT TGA AAG CA | Common | A | Common | ||
48069 | TCC AGA CTA AAA GGA AAC ATG C | Wild type Reverse | A | Wt R | ||
48071 | Fluorophore-1 | ACT GTG GTA ATT GAG TAT CTA GTC AAT CC | Quencher-1 | WT Probe | ||
48072 | Fluorophore-2 | TGA AAC GCC AAA CTG TTA GGA | Quencher-2 | MUT Probe | ||
49050 | ATG TTC AGC ATG TCC ATC GT | Mutant Reverse | A | Mut R |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48068 | 0.40 uM |
48069 | 0.40 uM |
49050 | 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 |