User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/02: Difference between revisions
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* Gibson assembly: order primers for vector pSB1A3 | * Gibson assembly: order primers for vector pSB1A3 | ||
* Golden Gate assembly: order primers for pSB1A3 and Brady's parts | * Golden Gate assembly: order primers for pSB1A3 and Brady's parts | ||
* Order enzyme for Golden gate assembly: | * Order enzyme for Golden gate assembly: BsmBI | ||
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'''Gibson Assembly design''' | '''Gibson Assembly design''' | ||
* Previously tried to ligate Gibson insert into X/S cut pSB1A3, unsuccessful. This time, PCR everything, including the vector, then do Gibson assembly. | * Previously tried to ligate Gibson insert into X/S-cut pSB1A3, unsuccessful. This time, PCR everything, including the vector, then do Gibson assembly. | ||
* Retry assembly "hPCD-BL01" (see [http://openwetware.org/wiki/Haynes_Lab:Notebook/Engineering_PC-TFs/2012/12/07]). Make sure primer sequence overlaps with Brady's external sequences: | * Retry assembly "hPCD-BL01" (see [http://openwetware.org/wiki/Haynes_Lab:Notebook/Engineering_PC-TFs/2012/12/07]). Make sure primer sequence overlaps with Brady's external sequences: | ||
** BBP-hPCD fwd: '''gaattcgcggccgcttctaga'''-tggagctttcagcggtggg (first 21 = BioBrick prefix) | ** BBP-hPCD fwd: '''gaattcgcggccgcttctaga'''-tggagctttcagcggtggg (first 21 = BioBrick prefix) | ||
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'''Golden Gate design''' | '''Golden Gate design''' | ||
* Use '''BsmBI''', per Dave Savage's recommendation | |||
** cgtctc - 5bp overlap - part - 5bp overlap - gcagaga | |||
** Forward - 5'-cgtctc NNNNN+20bp TOP | |||
** Reverse - 5'-cgtctc nnnnn+20bp rev comp | |||
New primers | |||
# gg_BBS F: 5'-'''cgtctc'''actagtagcggccgctgcag (should also work for V0120) | |||
# gg_BBP R: 5'-'''cgtctc'''tctagatgcggccgcgaattc (should also work for V0120) | |||
# gg_BBP-hPCD F: 5'-'''cgtctc'''CTAGAtggagctttcagcggtggg | |||
# gg_hPCD-BL01 R: 5'-'''cgtctc'''TCCATtctttccctttcctcaaagg | |||
# gg_ | |||
Revision as of 18:52, 2 January 2013
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01/02/13
Gibson Assembly design
New primers designed to amplify pSB1A3 backbone (should also work for V0120)
Golden Gate design
New primers
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