Daniel:Notebook/ComboLock/2016-8-16
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Positive Control Amplicon Production
Theory
The C1 and C3 amplicons
C1 Amplicon (PCAmp1): 5-ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG-3
C2 Amplicon (PCAmp2): 5-GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/-3
C3 Amplicon (PCAmp3): 5-ACGGCGGACCTCGCACGGCTGCCAACCCGTGGACGGTCGCGTTCGATGCGTC-3
Reaction Trimers:
LatchX2-Rev 3-GGCACCTGCCAGCGCAAGTGATTTAC CGGGCATAGCCGCTACGCACTAGCCC-5 PCAmp1|PCAmp2 5-ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG|GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/
LatchX3-Rev 3-GGCACCTGCCAGCGCAAGCTACGCAG CGGGCATAGCCGCTACGCACTAGCCC-5 PCAmp3|PCAmp2 5-ACGGCGGACCTCGCACGGCTGCCAACCCGTGGACGGTCGCGTTCGATGCGTC|GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/
Protocol
To complete the ligation reaction it is necessary that the oligo on the 3' end of the amplicon have a phosphate group on its 5' end. This requires a T4 Polynucleotide Kinase reaction (or ordering an oligo with a 5' phosphate). After the addition of the 5' phosphate I will incubate the oligos together in equal molar ratios to and add ligase to perform the ligation reaction.
- Phosphorylation