What concentration of tetracycline should be used for selecting the LMG194 strain?

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The LMG194 strain can be selected on LB plates containing 25 ug/mL tetracycline.

Answer Id: 3251

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504c296f8eb5fd521e744da4e8371f28_FAQ

What is the difference between vectors that contain a pUC origin and vectors that contain a pBR322 origin?

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Both pBR322 and pUC vectors have the same origin of replication. The locus that controls copy number is the "rop" locus; in pUC vectors, the rop locus is mutated to allow for much higher copy numbers. Therefore plasmids containing the pUC origin produce a high copy number of the plasmid. Most Invitrogen™ vectors have origins of replications that are pUC-derived and yield a high amount of DNA in E. coli. Plasmids derived from pBR322 will yield a lower plasmid copy number than plasmids derived from pUC. pBR322 gives about 15-20 copies per cell, while pUC gives about 500-700 copies.

Answer Id: 3247

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ac45088df2e8d3cd2d8fbafceb920878_FAQ

What is the molecular weight of the L-arabinose included in the pBAD kits?

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The molecular weight of arabinose is 150.1 g/mol.

Answer Id: 3249

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0613239e122094abb4ef998c01d16958_FAQ

What concentration of tetracycline should be used for selecting the LMG194 strain?

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The LMG194 strain can be selected on LB plates containing 25 ug/mL tetracycline.

Answer Id: 3250

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326fb04c3abf030fe3f4e341f39b573f_FAQ

What is the purpose of the LMG194 strain provided in the pBAD Kits?

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The LMG194 strain is provided to allow for growth of your plasmid construct under conditions of maximal repression. This strain is capable of growth on minimal media that includes glucose as the sole carbon source. Glucose provides maximal repression of the pBAD promoter.

Use of the LMG194 strain is not absolutely necessary since the TOP10 strain may also be used for expression. Note: all suitable strains must have mutations in the ara gene locus to prevent the breakdown of arabinose when it is added to the medium.

Answer Id: 3252

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1e3b21cb226c39b6aa4634f820b372f3_FAQ

What media should be used to grow the LMG194 strain? Will LMG194 grow in M9 alone?

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LMG194 will grow in LB and RM medium that contains M9 salts. LMG194 will not grow in M9 salts alone. RM medium is used to ensure low basal expression levels from the pBAD promoter.

Answer Id: 3253

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4ad13f04ef4373992c9d3046200aa350_FAQ

What strains of E. coli should be used with the pBAD inducible promoter system?

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Any E. coli strain that is araBADC- and araEFGH+ is suitable for use with the pBAD promoter. Suitable strains that can be used include TOP10, TOP10F', and DH10B. Cells that are not araBADC- and therefore cannot be used with pBAD constructs, include DH5alpha, OmniMAX, Mach1, BL21(DE3) and INValphaF'.

Answer Id: 3254

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8deb8d1dd92840f975b6931ab3a3c61e_FAQ

How should the pBAD promoter be induced when the cells are growing under maximal repression (i.e. in the presence of D-glucose)?

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If the construct is growing under maximal repression (i.e. in LMG194 with D-glucose in RM media), the cells should be harvested and resuspended in RM medium containing 0.2% glycerol and the appropriate concentration of arabinose (determined empirically). If cells are growing in LB medium, then arabinose may be added directly to the medium. Note that TOP10 cells will not grow in RM medium.

Answer Id: 3255

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2067e2650cd701ae71c68080f9dbbdc1_FAQ

Has Invitrogen™ mapped the exact transcriptional start site of the pBAD promoter?

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No, the exact start site of the pBAD promoter has not been determined experimentally.

Answer Id: 3256

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2131f8ecf18db66a758f718dc729e00e_FAQ

What is the purpose of the -35 and -10 consensus region, or sequence, that is found in prokaryotic promoters?

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In prokaryotes, such as E. coli, the promoter consists of two short regulatory sequences located 10 and 35 nucleotides upstream of the gene, respectively. The sequence located at -10 is called the Pribnow box (consensus sequence: TATAAT) and is absolutely essential for the transcription initiation. The sequence at -35 (consensus sequence: TTGACA) facilitates high transcription rate. Both regions are recognized by the sigma subunit of RNA polymerase, and instruct the holoenzyme where to start transcription. Once polymerization begins, the sigma subunit dissociates, and the core enzyme continues to transcribe the DNA template.

Answer Id: 3257

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4ea83d951990d8bf07a68ec3e50f9156_FAQ

What is the purpose of the RBS sequence found in prokaryotic vectors?

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The RBS, which stands for Ribosomal Binding Site, is required for translational initiation in E. coli, and consists primarily of purines. The consensus core region, or Shine-Dalgarno sequence (AGGAGG) is frequently located 8-12 base pairs upstream of the initiating codon AUG. This sequence forms base pairs with a complementary sequence located at the 3' end of the 16S rRNA molecule of the ribosome and assists in the recognition of the proper AUG of translation initiation.

Answer Id: 3258

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485843481a7edacbfce101ecb1e4d2a8_FAQ

What protein yields should I expect to get with the pRSET expression vectors?

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We've expressed CAT and Beta-Gal in volumes from 2 ml to 50 ml and get about 20-50 µg protein/ml of culture. Protein yield is dependent on the type of protein being expressed and the culturing conditions.

Answer Id: 3259

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a87d27f712df362cd22c7a8ef823e987_FAQ

Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?

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A. TA Cloning:
- This cloning method was designed for pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 of 15:1 ratio of Taq to proofreader polymerase will still generate enough 3’ A overhangs for TA cloning
- The recommended Life Technologies® polymerases include Platinum® Taq, AccuPrime™ Taq, Platinum® or AccuPrime™ Taq High Fidelity, AmpliTaq®, AmpliTaq Gold®, or AmpliTaq Gold® 360.

B. Blunt cloning:
- Use proofreading enzymes such as Pfx50, Platinum® or AccuPrime™ Pfx.

C. Directional TOPO cloning:
- Pfx50 or AccuPrime Pfx work well.

Answer Id: 6651

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06ccc6fac98a777fce43a972eaca83df_FAQ

What are the requirements for primer design when using directional TOPO® cloning?

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Please consider the following when designing your primers:

- The 3’ pcr primer cannot contain homology to the 5’ flap sequence GTGG.
- The enzyme you use must create a blunt-ended PCR product for cloning.
- Primers cannot contain 5’ phosphates, which will block the 5’ OH nucleophile reactive group.
- The reading frame must be considered when you are designing your primers.

Answer Id: 6738

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ddd993b2fef3fdff101872bb03cfded8_FAQ

Can I use a Taq polymerase to generate my gene of interest for directional TOPO® cloning?

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No, your gene of interest must be amplified with a proofreading polymerase such as AccuPrime™ Pfx or Platinum® Pfx that leaves blunt ends for directional TOPO® cloning.

Answer Id: 6743

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