Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:33260679-33260761 83bp GGAGATTGGGCCCCTTTAG TGGCAAAAGGAAAGACCTGA
Mut= 85 bp
Wt= 83 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TTCTCGTGGCACGTCCGCCTGAGTCGCATGGCAATTGATAGCATATTTCAGCATGAGGCACTGGGGAGAAGAGACTGGCTAGGGCTTTGGAGTGACGCTCCTGGGGACactctcTGAGCCCTGTGCTGTATTTgccctcacagagatctatgttgggtgtgaaatgaaggagctagtgagcatcccgtggccagtggacgaggtggctctgaggctgactcctgggagattgggcccctttagcacctgtacttctatccagggtttgatagGTTGTGGgcatacattcaggtctttccttttgccatttgtcatagcccctggcctgtcagcttttctgattgattttttcccctccagataaatttcttttcattccagcttttgaatcgaaaagacaagactacttttgagaaacttgactacctgatgtccaaagaagacaactacaagcgaacccgggactacatccgaagtctgaagatggtgcccagcattccctatctaggtatgagtctcagttattttattattaactttccaggcaacataacatgctcagggccaaataagatgtttttacagtttctagttgagatgtttccaggaagcagcagggatcatggataaagaggtggtttggttggctccttggttggagaagGCTGTTGATGGTTCCCAGGATAGGAACACTAGTGaggtgGAAACAAGGCAACTCTGTCTTATGTACCTGGACTCTGAGGGCCCGGGCTACCGAGCTGTGAGGGCTCCAGGCATCCAGGCTGCAGCCCAGATAGGTCATGATGTTGGTAGGTCATGCTGTCT
This mutation is a 524 bp deletion beginning at Chromosome 2 position 33,260,322 bp for 139 bp then a 7 bp (GTTGTGG) endogenous retention followed by an additional 385 bp deletion ending after 33,260,852 bp (GRCm38/mm10). In addition, there is a 6 bp deletion (ACTCTC) 19 bp before the 524 bp deletion as well as a 5 bp deletion (GTGGAG) 31 bp after the 524 bp deletion, neither of which are expected to alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41338 | GGA GAT TGG GCC CCT TTA G | Wild type Forward | A | |||
41339 | TGG CAA AAG GAA AGA CCT GA | Wild type Reverse | A | |||
41340 | ACT GGC TAG GGC TTT GGA GT | Mutant Forward | A | |||
41341 | CCT ATC CTG GGA ACC ATC AA | Mutant Reverse | A | |||
41342 | Fluorophore-1 | TTT GAT AGG TTG TGG GCA TAC A | Quencher-1 | WT Probe | ||
41343 | Fluorophore-2 | TGC TGT ATT TGT TGT GGG CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41338 | 0.40 uM |
41339 | 0.40 uM |
41340 | 0.40 uM |
41341 | 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 |