Available Vectors: Difference between revisions
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''' | '''A Note on Propagation and Purification''' | ||
Before listing the available part and destination vectors, a quick note on propagation and purification. Medium- and high-copy vectors (pFL series, pJT series, pDestET, pDestBBR) can be propagated normally in E. coli, using typical antibiotic concentrations. For plasmid purification, we recommend growing a single colony of plasmid-bearing E. coli in 10 mL LB + appropriate antibiotic overnight and miniprepping the next morning. | |||
For the BAC-derived destination vectors, however (pDestBAC, pDestRmceBAC, pDestPBBAC), we recommend a different approach. These BACs are derived from the pETcoco-1 vector from Novagen, which is a BAC with an arabinose-inducible medium-copy origin of replication. Growth of E. coli bearing these vectors should be carried out on LB plates containing no more than 10 ug/mL chloramphenicol. To purify these vectors, we recommend growing up a single colony overnight in 10 mL LB + 10 ug/mL chloramphenicol, diluting this 100x into 80 mL LB + 10 ug/mL chloramphenicol + 1.0% arabinose in a 500 mL flask, and growing with 200 rpm shaking at 30C for 24 h. Pellet the culture and follow the high-yield protocol for the Qiagen plasmid plus midi-prep kit, eluting in 100 uL of TE. This should provide ~ 100 ug plasmid DNA. | |||
'''All Vectors''' | '''All Vectors''' | ||
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'''Part Vectors''' | '''Part Vectors''' | ||
The pFL and pJT series of part vectors are described in [http://nar.oxfordjournals.org/content/42/1/681.long Torella et al. 2014]. Vector maps are provided in GenBank format below: | The pFL and pJT series of part vectors are described in [http://nar.oxfordjournals.org/content/42/1/681.long Torella et al. 2014]. We recommend using the pJT vectors when assembling into pDestET and pDestBAC destination vectors, and the pFL vectors when assembling into pDestRmceBAC and pDestPBBAC destination vectors. Vector maps are provided in GenBank format below: | ||
[[Media:pJT_U1U2 (pJT170).gb]] | [[Media:pJT_U1U2 (pJT170).gb]] (AmpR; 40 copies/cell) | ||
<br>[[Media:pJT_U2U3 (pJT172).gb]] | <br>[[Media:pJT_U2U3 (pJT172).gb]] (AmpR; 40 copies/cell) | ||
<br>[[Media:pJT_U3U4 (pJT174).gb]] | <br>[[Media:pJT_U3U4 (pJT174).gb]] (AmpR; 40 copies/cell) | ||
<br>[[Media:pJT_U4U5 (pJT176).gb]] | <br>[[Media:pJT_U4U5 (pJT176).gb]] (AmpR; 40 copies/cell) | ||
[[Media:pFL_U1U2.gb]] | [[Media:pFL_U1U2.gb]] (AmpR; 500 copies/cell) | ||
<br>[[Media:pFL_U2U3.gb]] | <br>[[Media:pFL_U2U3.gb]] (AmpR; 500 copies/cell) | ||
<br>[[Media:pFL_U3U4.gb]] | <br>[[Media:pFL_U3U4.gb]] (AmpR; 500 copies/cell) | ||
<br>[[Media:pFL_U4U5.gb]] | <br>[[Media:pFL_U4U5.gb]] (AmpR; 500 copies/cell) | ||
<br> | <br> | ||
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The pDestET and pDestBAC vectors are used in [http://nar.oxfordjournals.org/content/42/1/681.long Torella et al. 2014]. The pDestRmceBAC and pDestPBBAC are used in [http://nar.oxfordjournals.org/content/41/21/9967.long Lienert et al. 2014]. The pDestBBR vector is described in a forthcoming manuscript. | The pDestET and pDestBAC vectors are used in [http://nar.oxfordjournals.org/content/42/1/681.long Torella et al. 2014]. The pDestRmceBAC and pDestPBBAC are used in [http://nar.oxfordjournals.org/content/41/21/9967.long Lienert et al. 2014]. The pDestBBR vector is described in a forthcoming manuscript. | ||
[[Media:pDestET.gb]] | [[Media:pDestET.gb]] (AmpR; 40 copies/cell; ColE1 origin; good for strong bacterial expression) | ||
<br>[[Media:pDestBAC.gb]] | <br>[[Media:pDestBAC.gb]] (CamR 10 ug/mL; 1 copy/cell; BAC origin; amplifiable to 40/cell with arabinose; good for stability and decreased leaky gene expression) | ||
<br>[[Media:pDestRmceBAC.gb]] | <br>[[Media:pDestRmceBAC.gb]] (CamR 10 ug/mL; 1 copy/cell; BAC origin; amplifiable to 40/cell with arabinose; good for site-specific integration into mammalian genome) | ||
<br>[[Media:pDestPBBAC.gb]] | <br>[[Media:pDestPBBAC.gb]] (CamR 10 ug/mL; 1 copy/cell; BAC origin; amplifiable to 40/cell with arabinose; good for random integration into mammalian genome) | ||
<br>[[Media:pDestBBR.gb]] | <br>[[Media:pDestBBR.gb]] (KanR; broad host range BBR origin) | ||
<br> | |||
'''Obtaining Part and Destination Vectors''' | |||
Part and Destination vectors can be obtained from the Silver Lab. E-mail [mailto:jtorella@fas.harvard.edu jtorella@fas.harvard.edu] to request them, or for more information. | |||
<br> | |||
Latest revision as of 09:52, 3 May 2014
A Note on Propagation and Purification
Before listing the available part and destination vectors, a quick note on propagation and purification. Medium- and high-copy vectors (pFL series, pJT series, pDestET, pDestBBR) can be propagated normally in E. coli, using typical antibiotic concentrations. For plasmid purification, we recommend growing a single colony of plasmid-bearing E. coli in 10 mL LB + appropriate antibiotic overnight and miniprepping the next morning.
For the BAC-derived destination vectors, however (pDestBAC, pDestRmceBAC, pDestPBBAC), we recommend a different approach. These BACs are derived from the pETcoco-1 vector from Novagen, which is a BAC with an arabinose-inducible medium-copy origin of replication. Growth of E. coli bearing these vectors should be carried out on LB plates containing no more than 10 ug/mL chloramphenicol. To purify these vectors, we recommend growing up a single colony overnight in 10 mL LB + 10 ug/mL chloramphenicol, diluting this 100x into 80 mL LB + 10 ug/mL chloramphenicol + 1.0% arabinose in a 500 mL flask, and growing with 200 rpm shaking at 30C for 24 h. Pellet the culture and follow the high-yield protocol for the Qiagen plasmid plus midi-prep kit, eluting in 100 uL of TE. This should provide ~ 100 ug plasmid DNA.
All Vectors
A zip-file containing genbank files of all basic, currently-available part and destination vectors is available here: Media:All_Vectors.zip.
Part Vectors
The pFL and pJT series of part vectors are described in Torella et al. 2014. We recommend using the pJT vectors when assembling into pDestET and pDestBAC destination vectors, and the pFL vectors when assembling into pDestRmceBAC and pDestPBBAC destination vectors. Vector maps are provided in GenBank format below:
Media:pJT_U1U2 (pJT170).gb (AmpR; 40 copies/cell)
Media:pJT_U2U3 (pJT172).gb (AmpR; 40 copies/cell)
Media:pJT_U3U4 (pJT174).gb (AmpR; 40 copies/cell)
Media:pJT_U4U5 (pJT176).gb (AmpR; 40 copies/cell)
Media:pFL_U1U2.gb (AmpR; 500 copies/cell)
Media:pFL_U2U3.gb (AmpR; 500 copies/cell)
Media:pFL_U3U4.gb (AmpR; 500 copies/cell)
Media:pFL_U4U5.gb (AmpR; 500 copies/cell)
Destination Vectors
The pDestET and pDestBAC vectors are used in Torella et al. 2014. The pDestRmceBAC and pDestPBBAC are used in Lienert et al. 2014. The pDestBBR vector is described in a forthcoming manuscript.
Media:pDestET.gb (AmpR; 40 copies/cell; ColE1 origin; good for strong bacterial expression)
Media:pDestBAC.gb (CamR 10 ug/mL; 1 copy/cell; BAC origin; amplifiable to 40/cell with arabinose; good for stability and decreased leaky gene expression)
Media:pDestRmceBAC.gb (CamR 10 ug/mL; 1 copy/cell; BAC origin; amplifiable to 40/cell with arabinose; good for site-specific integration into mammalian genome)
Media:pDestPBBAC.gb (CamR 10 ug/mL; 1 copy/cell; BAC origin; amplifiable to 40/cell with arabinose; good for random integration into mammalian genome)
Media:pDestBBR.gb (KanR; broad host range BBR origin)
Obtaining Part and Destination Vectors
Part and Destination vectors can be obtained from the Silver Lab. E-mail jtorella@fas.harvard.edu to request them, or for more information.
