Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:40229653-40229772 120bp TGCCACTGATTTGAACAGGA TGGCTTCACAAAGTCCACAGT
Mut= 100 bp
Wt= 120 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
ATTCTTCAAAGCACATCTCCAGTGACTCACTTCTCCCAGAGAGGGTACAGTTCTCAGTTGCTCCTTCTGTATGAACTCAATGTGCTGCCAGTGGTGACGCCAGGACTCGAAGGATGTGGTCACCAGGTcccgcagggtgtggtcacctctaaagactgtcaccagctggtgtccaagcctgcaacctgtcagcctcatggtctggctccccagactgtccagtgactgaggccatttgcagatggttttcagttcccttgccactgatttgaacaggattcccatgattttgacttcaaagcatttttatgttggatttgcttaagaaatccccatttctcttttctttttcaggttactgtggactttgtgaagccatcgaaagttgggactttcaaaaaattgaaaagttagaggaacacagattacttttaagacgtttagaaccagaatttaaggccacagttgatccaaatgatatcctttctgaactatccgaatgtttgattaatcaggaatgtgaagaaatcagacaggtaaaccaatgccaggtactaaatttgaagaaaaatgcagagacattggaaatgcccatttttctgtcttgttttaggcccaaggataattgaaacccataaaagctctcaTCTAgcagatataatgactagaatagaatttttaaagtgaatggggtaatttttgtgctagactattagaaaattatttaacctatttgcagttaaagttgcccccttactttaaaaaaaatagtggtttatgcataatgcaaatcacaccaaacagtgcaacaattaaAAGGAAAAATATGTCAGGCTCTTGGGCATAGATACATTTATTACAGTCTCGCAGTCACTTAACTAGTGATGTGATGCCAGGCAGTTCTCTAAGCATCTGTGGGGTTTTTGTTGTTGTTGTTGTTGTTGTTTGTTTGT
This mutation is a 688 bp deletion beginning at Chromosome 4 position 40,229,215 bp for 523 bp followed by a 4 bp endogenous retention (TCTA) then a further 165 bp deletion ending after 40,229,902 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41168 | CAG GAC TCG AAG GAT GTG GT | Mutant Forward | A | |||
41170 | CAC TAG TTA AGT GAC TGC GAG ACT G | Mutant Reverse | A | |||
43047 | TGC CAC TGA TTT GAA CAG GA | Wild type Forward | A | |||
43048 | TGG CTT CAC AAA GTC CAC AGT | Wild type Reverse | A | |||
43049 | Fluorophore-1 | CCA TGA TTT TGA CTT CAA AGC A | Quencher-1 | WT Probe | ||
43050 | Fluorophore-2 | TGT CAG GCT CTT GGG CAT AGA TAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41168 | 0.40 uM |
41170 | 0.40 uM |
43047 | 0.40 uM |
43048 | 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 |