Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/26: Difference between revisions
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For assembly 1, amplify H2B with | For assembly 1, amplify H2B with | ||
1."1A3/H2B fwd", 5'- tctggaattcgcggccgcttCTAGA-ATGCCAGAGCCAGCGAAGTC | |||
2."H2B/LOV rev", 5'-CGTTCAAGTGTAGTAGCCAA-CTTAGCGCTGGTGTACTTGG | 2."H2B/LOV rev", 5'-CGTTCAAGTGTAGTAGCCAA-CTTAGCGCTGGTGTACTTGG | ||
…and amplify LOV with | …and amplify LOV with | ||
1."H2B/LOV fwd", 5'-CCAAGTACACCAGCGCTAAG-TTGGCTACTACACTTGAACG | |||
2."LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT | 2."LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT | ||
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For assembly 2, amplify LOV with | For assembly 2, amplify LOV with | ||
1."1A3/LOV fwd", 5'-tctggaattcgcggccgcttCTAGA-TTGGCTACTACACTTGAACG | |||
2."LOV/H2B rev", 5'-GACTTCGCTGGCTCTGGCAT-AAGTTCTTTTGCCGCCTCAT | 2."LOV/H2B rev", 5'-GACTTCGCTGGCTCTGGCAT-AAGTTCTTTTGCCGCCTCAT | ||
…and amplify H2B with | …and amplify H2B with | ||
1."LOV/H2B fwd", 5'-ATGAGGCGGCAAAAGAACTT-ATGCCAGAGCCAGCGAAGTC | |||
2."H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG | 2."H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG | ||
Revision as of 17:39, 8 December 2012
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November 26, 2012Gibson assembly written by Dr. Haynes
The Gibson assembly method joins double stranded fragments that have overlapping matching ends. Matching ends (instead of BioBrick cut sites) are artificially added via PCR. To do Gibson assembly, we need primers that span 40 bp across junctions of the individual parts.
We will cut the vector with XbaI and SpeI so the ends will look like this: pSB1A3 left end: …tctggaattcgcggccgctt/ pSB1A3 right end: /ctagtagcggccgctgcagt…
1. pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end …to get a circular plasmid.
2. pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
…and amplify LOV with 1."H2B/LOV fwd", 5'-CCAAGTACACCAGCGCTAAG-TTGGCTACTACACTTGAACG 2."LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT After PCR of each part, the two PCR products will be mixed with the X/S cut vector and subjected to isothermal assembly: http://openwetware.org/wiki/Haynes:Gibson_Assembly
…and amplify H2B with 1."LOV/H2B fwd", 5'-ATGAGGCGGCAAAAGAACTT-ATGCCAGAGCCAGCGAAGTC 2."H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG
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