Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:5886560-5886662 103bp GGCCAAGCAAGTGCATTTTA GATCCCAAACAGCGGAATC
Mutant= 100 bp
Wild Type = 103 bp
Wt Sequence (deletions in lower case):
TTAGAAATCATAAGCAGTCAGTCTCCAACAGGCATTGTAGGCCAAGCAAGTGCATTTTATTTAAGAGGGAGACTGCATGCAGAGCGCCCTTGTgttccactaagtgttttatgtgatgtttcagattccgctgtttgggatcatgtcatcggactctgcagatcccttctactggatgagagttattcttgcatccaacagaggtatgcacttctgacctccatcctgccaccaacctccatcctgccaccaacctccatcctgccaccaacctccatcctgccaccaaacagctgctttgtggttttaTATAGGAGAGACAAAACAAACCCATTGCTTAGGAGGAGAATCCAGCTTGTAATAATAACAACACTGGCGTGTGTGTGTGTGTGTG
This is a 216 bp deletion beginning at Chromosome 2 position 5,886,393 bp and ending after 5,886,608 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46738 | GGC CAA GCA AGT GCA TTT TA | Common | A | |||
46739 | GAT CCC AAA CAG CGG AAT C | Wild type Reverse | A | |||
46740 | GCT GGA TTC TCC TCC TAA GCA | Mutant Reverse | A | |||
46741 | Fluorophore-1 | AGC GCC CTT GTT ATA GGA GAG | Quencher-1 | MUT Probe | ||
46742 | Fluorophore-2 | CCA CTA AGT GTT TTA TGT GAT GTT TC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46738 | 0.40 uM |
46739 | 0.40 uM |
46740 | 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 |