Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr7:16076026+16076137 112bp CGAGTGCTTTTGGTTTGGA TGGATCAGGTTGAGCTTCTAGG
Mut= 96 bp
Wt= 112 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
AGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTCTCTCTCTCTGTGTGTGTGTGTGAGAGAGAGACACACAGAGAGAGAGAGACAGAGAGAGAGCGAGTAAATAAAGCACTCACCTCCTCTAAGCTCTCTAATCAGATATAAGGTTGTTTCTAACATTTTACTAGAAAAAGCAGGTAGATTTTTTTTttTTATAAAAAGGTGAGAACCCAAGAGCAGAGCAAGATGGCGAGTGCTTTTGGTTTGGATAATGCATCTCTAGATAGACATTGTATGGGTAGGTTGGCTTctacccctgcttgtcttatcatactgctaccctagaagctcaacctgatccatacctgcacatcttgcaccaagacctggtgcagttctatagataaggcctggtccagacctcagctaagccttgtcgtctggccgtgcagagcctctgaggtgggggagtctcagactactgggacagggctctgtgctgagggtctatattgctttagcactcccctatcctatctcagcataacctgtgctttctccctttgtttagacctggggctgggcgcactcaaggtgcctaaagaggctgatgaaggaggcagggccactgggagtacaaggaaaggcaagcgacagcacagttcccctcagaatccactcctggactgcagcctgtgtgggaaagtgttcagcagcgccagttccctgagcaagcattacctgacgcacagccaggagagaaaacacgtgtgcaaagtctgcagcaaagccttcaagcggcaggaccacctgtatgtcaagaatctggccagaagggcagggggcatgcacctgtcctcctaccctctcccccatccatctggtcctctgttgagttcccacttctgtgcttctcagatgtatgtcccctgtcctccagtgtggactctgttctCTTCACACTGCTATTGTCTGGGGGCAGTCTCTTTACTAAGCCCCAAATTTACACCAGTGAGGAAATCCCTGTCTATTTGCAGACATGGCTCTTGTGGTTTCTAAATGTGATGAGTTACCAGGCGTGCTGGCACACTGGGGAGGTAGAAGCAGGAGGATCAAGAGTCTAAGGCCAGCCTCAACTACATACATTACAAGTTTAAAGCCCACC
This mutation is a 645 bp deletion beginning at Chromosome 7 position 16,076,086 bp and ending after 16,076,730 bp (GRCm38/mm10). In addition, there is an additional 2 bp deletion (TT) 98 bp before the 645 bp deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48990 | GTA AAG AGA CTG CCC CCA GA | Mutant Reverse | A | |||
48991 | CGA GTG CTT TTG GTT TGG A | Common | A | |||
50047 | TGG ATC AGG TTG AGC TTC TAG G | Wild type Reverse | A | |||
50048 | Fluorophore-1 | CCC TGC TTG TCT TAT CAT ACT GCT A | Quencher-1 | WT Probe | ||
50049 | Fluorophore-2 | AGG TTG GCT TCT TCA CAC TGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48990 | 0.40 uM |
48991 | 0.40 uM |
50047 | 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 |