Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:3595585-3595734 150bp ACAATAGCGCTTCTCACAGG TCTGCCTAAAGACTACCACACTCC
Mut = 116 bp
Wt = 150 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATACCGGGAGGGCCGACGCCCTCGCGCGGTCAAGGTAGAGTGACTCGGATTCTTCTCTCCCCTCGGCAGGGACACTAAATTCCTGTGAGCACTAGCTCCCCAAGGCCTGCCTCCATAAAGGCATGGCAGGGATGTTGTGAGATCCATCTCTAAGTGGAGGGGGCACGAAGAACCCGGGCCCCGGTACCAAAGGTTATCACTACCCTCGGTTAGCTGCTTACCAAACCTCCAACCCACTATTTTTAGCTTTGTATCCTATGTGTTTACAATAGCGCTTCTCACAGGGCaagcccagggtgagtgagtgttcagtggctgtttacattaacctctctggagtcagcttttggaaaagacgtctgttaagtggtttagtcagatacccttatgtggagtgtggtagtctttaggcagaagagtgacagtttagtgtgtatgttttgcagaccggattgtttcttgttttcaaagctccatttaaaagggggtgggaggaccgatttgccataggcttgtttgaagagtacatttaacgaggtctcctttgactttaggtatatacaatcaatttggagtctcagtacttactaatacaaggagttcctgcagtgggagccatgaaggaattagttgagcgttttgctctgtatggtgcgattgaacagtataacgctctagatgaatacccagcggaagattttacagaagtttatctcattaaatttgtgaaattacagagtgcaaggtatgtacaagttaagcagaactcctgttaaaatcttattcctgttgctgtcattttggccccaactgtttcccaaatgTACCCAAAGCACAGAAGCACTTCTTGCTCTCAGTCTTCCTGATTGGGGGGAGGAGGGGAGGGGACAGGGTTTATTTGGATAGCCTTAGCTGTGCTAGAATTAGTTCTGTAGACCAGGCTGGCCTTGAACTCAAAGATCCCTTTGCCTTTGCCTCTGACTCTCGAGTGCTGGGTTTAAAGGTATGCACCACTCCACCTCGCCTTCCAGTTTGTTTTTCACCAGGCCTCACATCTGCGACTTAATCACTCTAACAGTGA
This mutation is a 427 bp deletion beginning at Chromosome 5 position 3,595,181 bp and ending after 3,595,707 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49315 | ACA ATA GCG CTT CTC ACA GG | Common | A | |||
49316 | TCT GCC TAA AGA CTA CCA CAC TCC | Wild type Reverse | A | |||
51231 | Fluorophore-1 | CTT CCT GAT TGG GGG GAG G | Quencher-1 | MUT Probe | ||
51232 | Fluorophore-2 | AGG GTG AGT GAG TGT TCA GTG G | Quencher-2 | WT Probe | ||
51931 | GGC TAT CCA AAT AAA CCC TGT | Mutant Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
49315 | 0.50 uM |
49316 | 0.50 uM |
51931 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
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 |