Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:46717404-46717513 110bp AAGATCCAGAAGGATGAAAACA CACAACTGTTTCCAGCAAAATTAC
Mut= 95 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTCCACCAGCTGCACTAACCATATTTCTAGCTCTTTGTCTTCCTTAAAGAATGTGACACAAAATAATTGTTGAAATGACTTACAGTGTGTCTTTTAATGTATTAAGATCCAGAAGGATGAAAACATGTACTACCTACCACtaaatctagatcttggtataatgacataccattcacaaactgaatcttagtaattttgctggaaacagttgtgatttgcttggcatgactttctgtaagtacttacaaagataagttttctaaagtagtcttaatgtatgttttctccaacagggcattcttagcagggttacagaatctgttaagaatattgtaccagggtggctacaaagatacttcaacaagagtgaaaatgcttgcagctgttcaccagatgcagatgaagttccaccctggccagaaaatagagaagatgagcatgcgatttatgcggatgagaacacaaacactgatgatgggagaatcacccctgaccctgcaggcagtaacacagaaggtaaatactgtggatatttggttacaaagcgtagatcttcctaatttaaatatctttatattacctgagtgTGATAGCATCTGACCAAGTAATATATAAATCCTGTATGGAACATTTGGAATGAAGTTCACACTGAGTATGTTTATTGAGTAGTTTTTAAAAATATTTGTGTGTATATGCACATGGGCTGGCATGCCACAGTGTGTTGTGTGGAGATAAAGATCAA
This mutation is a 447 bp deletion beginning at Chromosome 13 position 46,717,030 bp and ending after 46,717,476 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40541 | AAG ATC CAG AAG GAT GAA AAC A | Common | A | |||
40542 | CAC AAC TGT TTC CAG CAA AAT TAC | Wild type Reverse | A | |||
40543 | GAA CTT CAT TCC AAA TGT TCC A | Mutant Reverse | A | |||
40544 | Fluorophore-1 | TAG ATC TTG GTA TAA TGA CAT ACC ATT CA | Quencher-1 | WT Probe | ||
40545 | Fluorophore-2 | ACC TAC CAC TGA TAG CAT CTG ACC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40541 | 0.40 uM |
40542 | 0.40 uM |
40543 | 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 |