What is the best molar ratio of PCR product:vector to use for TOPO™ TA cloning? Is there an equation to calculate the quantity to use?

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Answer

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO™ cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

Answer Id: E7648

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

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Answer

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

Answer Id: E3256

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What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

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Answer

You may have to try different ratios ranging from 1:1 to 15:1 insert:vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

Answer Id: E7645

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

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Answer

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: E3253

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What arabinose should I use for induction of my pBAD expression system?

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We recommend using L-arabinose to regulate expression of your gene when using the pBAD expression system. In the presence of L-arabinose, expression from pBAD is turned on while the absence of L-arabinose produces very low levels of transcription from pBAD (Lee, 1980 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1980+pbad]; Lee et al., 1987 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1987+arbinose]).

Answer Id: E9712

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

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Answer

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: E3257

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

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Answer

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: E3254

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Should I use TOP10 cells or the LMG194 E. coli strain you offer for expression with my pBAD system?

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Answer

Top10

Advantages:
- Saves time, can go directly from cloning to expression.
- The glycerol stock is more stable because these strains are endA- and recA-.

Disdvantages:
- This strain is not protease-deficient. Therefore, the protein may be degraded.

LMG194

Advantages:
- Grows well in minimal media, except M9.
-Have to transform the plasmid into the cells just for expression.
-RM medium with glucose to ensure low basal level of protein.

Disadvantages:
- Not protease-deficient. Therefore, the protein may be degraded.
- The glycerol stock may not be stable because this cell strain is not recA- or endA-.

Answer Id: E9717

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

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Answer

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

Answer Id: E3251

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Can you tell me the sequence of primers that can be used in pBAD vectors to sequence my gene of interest?

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Answer

The pBAD vectors contain a forward and reverse pBAD primer flanking the gene of interest. The sequences are as follows:

pBAD forward primer: 5'-ATGCCATAGCATTTTTATCC-3'

pBAD reverse primer: 5'-GATTTAATCTGTATCAGG-3'

Answer Id: E9715

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How does the regulation of the araBAD (pBAD) promoter work?

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Answer

The araBAD promoter (pBAD) used to control expression of T7 RNA polymerase in BL21-AI™ is both positively and negatively regulated by the product of the araC gene (Ogden et al., 1980; Schleif, 1992). AraC is a transcriptional regulator that forms a complex with L-arabinose. In the absence of L-arabinose, the AraC dimer contacts the O2 and I1 half sites of the araBAD operon, forming a 210 bp DNA loop (see figure below). For maximum transcriptional activation, two events are required.

- L-arabinose binds to AraC and causes the protein to release the O2 site and bind the I2 site that is adjacent to the I1 site. This releases the DNA loop and allows transcription to begin.
- The cAMP activator protein (CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2.

Please note, basal expression levels can be repressed by introducing glucose to the growth medium. Glucose acts by lowering cAMP levels, which in turn decreases the binding of CAP. As cAMP levels are lowered, transcriptional activation is decreased.

Answer Id: E9713

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

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Answer

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: E3258

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

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Answer

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

Blunt cloning:
- Use a proofreading enzyme such as Platinum™ SuperFi™ DNA Polymerase.

Directional TOPO cloning:
- Platinum™ SuperFi™ DNA Polymerase works well.

Answer Id: E6651

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

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Answer

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: E3247

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

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Answer

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: E6738

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