Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:89651669+89651787 119bp TGTCAAAGGGGCATAGCAGT CCTGGCAATCAGCATTGTA
Mut= 101 bp
Wt= 119 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
TTAAAGGACACTTGTCCTTGGTCTCAGCACAGCTGTTGAGTATATAACCCAGATGCTTTGTTTTGAAAGGAATCCATGGGTGGGCCTTTGGGTTTCAGGTGTCCTCTGAAGGCATCACTGTTTTCCCATCTTAAGTCATTATTTCATAAACGCATGGAGCACTGGGCTTCATGGATGTGAACAGGCAAGCCATGAGCAATGGAGCACTGGAAGGCTTATGCTCTgccaggggccctttgtggtatcttcatatattgttttattccatagatgacggggccagtgttcaggaagtgctgacagaaatcagtaaccagaaaacggtgcccaatatttttgtgaataaagtgcacgtgggtggatgtgaccgaactttccaggtaatgatggtcatcagacacggtcctcaacactgtgtgttcacattgcaatttaatccatgtcaaaggggcatagcagtggaaaggcacaggggctgggtatgggataatcctgaagatgcaagggaaatgagctTTTCCATCAATACCTGAGAAGTTATACAATGCTGATTGCCAGGCACAGGAGGGACATTCTTGGCCTGAAGTCAGAGCAGCTGTGGCCCTCATAGAACTGAGTCTACTACCACTCCTCTATGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGAGTGATCATGAGTCCTGTGAGGGATTCATGGTGGCTCAGGAGCAGCTCATGACTCATGGGG
This mutation is a 292 bp deletion beginning at Chromosome 6 position 89,651,453 bp and ending after 89,651,744 bp (GRCm38/mm10). In addition, there is a 23 bp TTACCAGGCCCTGCCCAAATTTC insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64680 | TGT CAA AGG GGC ATA GCA GT | Wild type Forward | A | |||
64681 | CCT GGC AAT CAG CAT TGT A | Common | A | |||
64682 | CCA TGA GCA ATG GAG CAC T | Mutant Forward | A | |||
64683 | Fluorophore-1 | CTG AAG ATG CAA GGG AAA TGA | Quencher-1 | WT Probe | ||
64684 | Fluorophore-2 | AAG GCT TAT GCT CTT TAC CAG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64680 | 0.40 uM |
64681 | 0.40 uM |
64682 | 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 |