Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:106799813+106799895 83bp TGACACTCAAGCTCCTGCAC TGATCACAGCACAGTTTCACC
Mut= 83 bp
Wt= 83 bp
Fam=Mut
Hex=Wt
Wt Sequence: (deleted region in lower case)
GTGGAGCGGTAAGTGAGCCTTGCTTTAACAGTGGGGCTGGATAGGGCTGGCAGAGCCTTGGTGGGTAAGCATTTGCCTCATAAGCTTGATAACCTGAATTCCATTCCAAGAACCCACAGAGAAGGAGAGAAAGAACTCCCCAAAGTTGTCCTCTGACCTCCACACTTGGGTTGTGACACTCAAGCTCCTGCACAAGTATGCCTCTCCCCCCCAAAGTgtgcaaacagggggaaatggtgaaactgtgctgtgatcacctgcctgccctgctgtgagagccccccttgtctcttcttctccaagggactcatggcaggtggtggttggcttgctcatgtcattatcatgttggctcaggacagacagggtcttaacttagcctttatgtttgtctttttaaacaggtgaaactgagcagtattctaaagattctcatggagaagaagctggggaggacctagaacgtaccgaggaggcgaagtggagaaacgcttcttttatgaggtagtgtgcagtgcctgtgaatttcatcagacaagttatgcttcccactctgtgagattaagtccagcatactctaatgtccctccaaaagctgatgtgaacattttatgcctttctGAGCTCTTGACTTTCCTTTCCTATCTCTGCTTTCACAACTTTTTTCTTTTTCTTTTTTTTTTTTTTCTTTTTTTGGTTTTTCGAGACAGGGTTTCTCTGTATAGCCCTGGCTATCCTGGAACTCACTTTGTAGACCAGGCTGGCCTCGAACTCAGAAATCCGCCTGCCTCTGCCTCCCAAGTGCTGGGAGTAAAGGCGTGCGCCACCACGCCCGGCTCACAACTTTTTTCATTCTAGACTTTTGTTGAGATGGTTC
This is a 404 bp deletion beginning at Chromosome 11 positive strand position 106,799,857 bp and ending after 106,800,260 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38172 | TGA CAC TCA AGC TCC TGC AC | Common | A | |||
38173 | TGA TCA CAG CAC AGT TTC ACC | Wild type Reverse | A | |||
38174 | GTT GTG AAA GCA GAG ATA GGA AAG | Mutant Reverse | A | |||
38175 | Fluorophore-1 | TGC AAA CAG GGG GAA ATG | Quencher-1 | WT Probe | ||
38176 | Fluorophore-2 | CCC CAA AGT GAG CTC TTG AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38172 | 0.40 uM |
38173 | 0.40 uM |
38174 | 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 |