Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:44909874-44909992 119bp TGTTTTGTGGATGAGGAGACAG CCTTCCAGCCTATCTGTTGC
Mut= 137 bp
Wt= 119 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TCTATTTCTGACGTATTGGTTGCAGGCGAGGTCTGGGTGCACAGATGTGACCCACAGTGTCTGCTAGTCTCTTTATTACCTATAGCAATGCAGGgtacacgaggGTGCTCACGATGCAGGGCCGAGCCAGCCTGCACATTTTGCCTGGCCCTGCCAGTTGTTTTGTGGATGAGGAGACAGAGTCTCTGGGGCAGCCAGGGCAGTTTCCAAGTGctttggggttgctctggctgcctatgtaatatcctggtcacgtggcaacagataggctggaaggtgcactggggccatgtagccctgactgccccacggtgtcccacagggacagcatggacagtgtgaagcagagtgccgccctatgcctactgcgactctacaaggcctcgcccgacttggtgcccatgggcgagtggacggcacgtgtagtgcacttgctcaatgatcagcacatggtgaggtgcccggccgccccaccctcaagtgcctgaagcccatgtcttgccttctgccctgacattccaacccagacctcctgtccccagtgtgtatgccctgcccagtttccaagaaaccttcttccttcctcgataactccttggctgcctgggactgtcctcatctctgatgcagtaccttaacaagatttgcctatttctgagcctagagagcccatagcaaggccagtggactgactgccttgcaaagactgaccccaccctagactcccattagcccaccctgctcctgactcctgagctgacttaccctgatgccagggctttcccagtgacacccagcatgaagtccctcacgcctgagctccttgcagggagtggtcacagctgctgtcagcctcatcacctgtctctgcaagaagaatccggatgacttcaagacctgtatctccctggctgtgtctcgcttaagccgggtgagtgtgctgtcacactggccgtgggagggtctggctcccctgtgtctgtgatatctgtctctcctcccttccctaaaaggcaagccagcctgtgcttagaagccaggactctgcattggcttcctcgggctgtgtgcccccaggcaaatggcttcacttctctttgcttcttttcctgtgtggttcaggggctggtgtcagtgcggtctgcagCATGGTTGCTGTGGTAAGGTGCTGACAGCTCCCCTGCCTGCTCTTCTTCCTCCTGTTTGTCTACACATCCCTCCTTTGCCTCCCACCACCTCCACCCTTGTCCCCAGAGCGCCGCCCCGAGCCTTCTGTGTGCCTGGCTCTCTATGTCT
This mutation is a 934 bp deletion beginning at Chromosome 7 position 44909004 bp and ending after 44909937 bp (GRCm38/mm10). In addition, there is a 10 bp deletion ( GTACACGAGG) 109 bp before the 934 bp del that will not alter the results of the exon deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37545 | CCT TCC AGC CTA TCT GTT GC | Wild type Reverse | A | |||
39982 | TGT TTT GTG GAT GAG GAG ACA G | Common | A | |||
39984 | Fluorophore-1 | CTG GCT GCC TAT GTA ATA TCC TG | Quencher-1 | |||
39985 | Fluorophore-2 | AGT GCA TGG TTG CTG TGG T | Quencher-2 | |||
40758 | GAG GCA AAG GAG GGA TGT GT | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37545 | 0.40 uM |
39982 | 0.40 uM |
40758 | 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 |