Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:28845674+28845766 93bp TGGATTCCCTTGTCATTCAG CCCTCCCCCACATAACACTC
Mut= 99 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCTGGCTTCCTGGGTAACCTCACTTGATGGGTCACTCCCTAACAGCAGCATTGAGTCCAGACTTGGTTTGTTGGGCTTAAAGCAGACTTCTAATTTCTGGATAAGTGTGATGTTGCTATGGTGGCAGATGTCTCTGTGACCTTGGGGAGAGAGCTAAGCTACCTTCTTACTGTCAAAGACCTGCATGTTTTCAGAGAATAAAGGCAAAAAAGACTGTTTGAAGTGTGGTTTCTACAAGCAGATGGACATTTTTCTTTTCCCTTTTGTATAATGGCATGAGTAAGTTTTCTAGTTACTGTGAAAAGGGAGAGAAAAAAAACATTGGAagcctggattcccttgtcattcagtcacaattgaccttttttgtgtgtgttagctatgtaggattgtttctcagcctgagtgttatgtgggggagggggccttggatagcggagttattatgaggttgccttatatatcatcagtttgtaaaagcaaatcctgcagatactatagggtagagggcagactcaccccccagctgaaagtactggcttagtgggctttaatttaatcataagatgctgatgtcgctccagcattttgtctttatgtcattgaatatatttgcattaaagtaatatttatttcatattctcttttagatttctacaataaatacagtcttaaaaatgtccagtatgaccaggtaagaggttgaagctatttttgattttcttagggtttctttgagtacaagctgtccatggaaggcagagcacattcgtgtcgggtgctgggaacaactcaaactgctaaggtcttgacttagcgcttagcctatgggactccctacctgcttgtatgcagactggatttccttccatttcggcactcaagctccctactgcaagacatagctggtttaggacattaggacatctgccatctctgaaaatgacttgagccaaaatacAGTATTTGGTCGTGTTAGTTCTTAACAGTAAAAATAAGGTCAACCAAAATGATGGGAGTAAATATTTGCTAGGATTTAGAGGGATACTGAAAAGGTATTCTAAATGCTTGTTGCATTGGAAACACTTGTAAAAGAGAAATTCAGAATTAGACTATGAGCAGTGAGCTGTGTAGTTGTAGTTGACTTTGCATAAGCAAAATGCAAAAATATGGAAGAATAGAGAACCACCAGCCTTTTCCATTTATTTGCTTTTGTGACAGTCTCATAGTTCTGCTGGCCTCA
This mutation is a 635 bp deletion beginning at Chromosome 2 position 28,845,670 bp and ending after 28,846,304 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49010 | TGG ATT CCC TTG TCA TTC AG | Wild type Forward | A | |||
49011 | CCC TCC CCC ACA TAA CAC TC | Wild type Reverse | A | |||
49012 | TTC TAG TTA CTG TGA AAA GGG AGA G | Mutant Forward | A | |||
49013 | ACT CCC ATC ATT TTG GTT GA | Mutant Reverse | A | |||
49014 | Fluorophore-1 | ACA ATT GAC CTT TTT TGT GTG TGT | Quencher-1 | WT Probe | ||
49015 | Fluorophore-2 | CAT TGG AAG TAT TTG GTC GTG TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49010 | 0.40 uM |
49011 | 0.40 uM |
49012 | 0.40 uM |
49013 | 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 |