Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:43443887+43443976 90bp GCACCCTAACATCCTCAGGT CATCTCCCTCCTTTCCCAAG
Mut= 88 bp
Wt= 90 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case; and insertions with carrots ^g^ (C insertion):
GGCGGGACCGAGCCTTGGACGGGAGGCTAGAACTTAGAGGCCCCGCAGCCTGGGTTAGAGATCCTTGAGCTAGCAGCCACccaacCTGTCGGGCACACACGGAGCCTTGCCTCCCCCAGGTTCGGCACCGACAGTCGGGACAAGTCATGGTGCTGAAGATGAACAAGCTCCCCAGTAACCGGAGCAACACGCTAAGGGAGGTGCAGCTGATGAACCGGCTCCGGCACCCTAACATCCTCAGGTGAACGGCCTAGCCTCTT^gctggacagcttgctgggcaggaggaaaagtcccttgggaaaggagggagatgcagtggagcccgggctcttacagcgtatcctttatcctccccatcacctttcaggttcatgggggtctgtgtgcaccaagggcagctgcacgcgcttacagaggtaagcattaagcataggccaggaaagagggaaggcaccacacccccaagtgagagtgtcagtgatcttggtgggtctgctgggaaattaaaaagaagccacctgcctgcccaggacctcaggacctcctccaactaaaggcttcctgaacttccctgtgtgcaaaccacttattagaatactcagctggagttagtgatggagatattttgtatctgggtccaatacaggagccactagctacacgtggaatagactaaaacaagagctggtgtaacaaaagaatgtctgattatatttaaatgtattggataacatagctgtacatagtcctactgtgagagacagggtagcctagtatataaatgggaaccttagagcacagacccaccaacccttttgacctttgtgagcctcctgagtgtcaactgtcttaggagtggcaggcacagagcaccgacattcacacaccttgtctttccccatgtcccagtatatgaatgggggcaccctggaacagctgctcagctccccagaacccctctcctggccagtcaggctccacctagccctggacattgcacaaggcctacggtacctacacgccaaaggtgtgtttcaccgagacctcacatccaaggtaggctggccaagacgggtggaacgattaaagagggtttgtgactgttaagttgtaattggcctgtggatattgacatgtaggtagtagatacgttaggatg^TCGGGGTGCCAAGGTGTTGGAAACTATTAAAGAATGGGGACAGCAAGAGGGCTGGGGAATTGAGTACTGAGTGAGGTGGTGATAATGAGGTGCATTGGAGAACTGGGAGGGAAGGT
This mutation is a 899 bp deletion beginning at Chromosome 4 position 43,443,924 bp and ending after 43,444,822 bp (GRCm38/mm10). In addition, there is a 5 bp deletion (CCAAC) 175 bp before the deletion and a single bp [C] insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42934 | GCA CCC TAA CAT CCT CAG GT | Common | A | |||
42935 | CAT CTC CCT CCT TTC CCA AG | Wild type Reverse | A | |||
42936 | CCT CTT GCT GTC CCC ATT CT | Mutant Reverse | A | |||
42937 | Fluorophore-1 | TTG CTG GGC AGG AGG A | Quencher-1 | WT Probe | ||
42938 | Fluorophore-2 | CTC TTC TCG GGG TGC CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42934 | 0.40 uM |
42935 | 0.40 uM |
42936 | 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 |