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K10101

Product FAQ

What is the optimal spacing between the ribosome-binding site (RBS) and the ATG when cloning into a bacterial expression vector?

Answer

The sequence between the RBS and ATG should be between 8-12 bp and not contain any palindromic sequence.

Answer Id: E9696

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Will carbenicillin in place of ampicillin help to increase expression levels from the pET TOPO™ vectors?

Answer

For most purposes, ampicillin works well for selection of transformants and expression experiments. However, degradation of ampicillin is enhanced during the late stages of bacterial growth, when the pH of the culture tends to decrease. This could result in non-selective conditions, resulting in the appearance of satellite colonies on plates, and if the transferred plasmid is unstable it may result in the loss of the plasmid and low expression levels. Carbenicillin is generally more stable under acidic conditions than ampicillin and studies have shown that using carbenicillin in place of ampicillin may help increase expression levels by preventing the loss of the pET TOPO™ plasmid. Use at a concentration of 50 ug/ml.

Answer Id: E3883

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Product FAQ

How can I make a glycerol stock of my desired construct?

Answer

Once you have obtained your desired construct, we recommend that you store your clone as a glycerol stock. Please follow these steps to create a glycerol stock:

- Grow 1 to 2 mL of the strain to saturation (12-16 hours; OD600 = 1-2) in LB containing 50-100 μg/mL ampicillin
- Combine 0.85 mL of the culture with 0.15 mL of sterile glycerol
- Mix the solution by vortexing
- Transfer to an appropriate vial for freezing and cap
- Freeze in an ethanol/dry ice bath or liquid nitrogen and then transfer to –80 degrees C for long-term storage.

Answer Id: E9727

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Product FAQ

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

Answer

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: E3259

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Product FAQ

My gene of interest is toxic to bacterial cells. Are there any precautions you can suggest?

Answer

Several precautions may be taken to prevent problems resulting from basal level expression of a toxic gene of interest. These methods all assume that the T7-based or Champion™-based expression plasmid has been correctly designed and created.

- Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase (i.e., DH5?™).
- If using BL21 (DE3) cells, try growing cells at room temperature rather than 37 degrees C for 24-48 hr.
- Perform a fresh transformation using a tightly regulated E. coli strain, such as BL21-AI™ cells.
- After following the transformation protocol, plate the transformation reaction on LB plates containing 100 μg/mL ampicillin and 0.1% glucose. The presence of glucose represses basal expression of T7 RNA polymerase.
- Following transformation of BL21-AI™ cells, pick 3 or 4 transformants and inoculate directly into fresh LB medium containing 100 μg/mL ampicillin or 50 μg/mL carbenicillin (and 0.1% glucose, if desired). When the culture reaches an OD600 of 0.4, induce expression of the recombinant protein by adding L-arabinose to a final concentration of 0.2%.
- When performing expression experiments, supplement the growth medium with 0.1% glucose in addition to 0.2% arabinose.
- Try a regulated bacterial expression system such as our pBAD system.

Answer Id: E9741

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Product FAQ

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

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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Product FAQ

How much IPTG can I use to induce expression from a T7 promoter containing bacterial expression vector?

Answer

This can vary somewhat, but we typically suggest a starting range of 0.1-5 mM IPTG.

Answer Id: E9697

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Product FAQ

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

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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Product FAQ

I am using a bacterial expression vector containing the T7 promoter. At what OD should I induce my cells?

Answer

The time of induction can vary widely. Successful experiments using the T7 systems have induced at an OD600 of 0.1-1.2. Generally speaking, induction at high ODs will lead to lower expression yields, as the cells will stop growing rapidly after the density is too high. The optimal OD600 for induction is 0.4 to 0.6.

Answer Id: E9698

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Product FAQ

What is the source of the EM7 promoter?

Answer

The EM7 promoter is a synthetic promoter derived from the T7 promoter. The promoter is typically used to drive expression of antibiotic resistance genes for selection in E. coli.

Answer Id: E3270

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Product FAQ

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

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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Product FAQ

What is the maximum size plasmid transformed for bacterial expression?

Answer

The largest size plasmid we've tried is 16 kb, but you should theoretically be able to transform a plasmid as large as 30 kb before efficiency begins to drop.

Answer Id: E9720

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Product FAQ

I am using a bacterial expression vector containing the T7 promoter. At what temperature should I perform my induction?

Answer

Induction can be performed at a variety of temperatures, ranging from 37 degrees C to 30 degrees C to room temperature. A lower temperature typically requires a longer growth time.

Answer Id: E9699

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Product FAQ

Does Thermo Fisher Scientific have a vector for co-expression of two proteins in E. coli?

Answer

A dual promoter vector is the best option for expressing two proteins at the same time. Unfortunately, we do not offer any prokaryotic expression vectors containing dual promoters. Another option is to use two different but compatible vectors at the same time. For example, you can try using a pET vector such as pET100/D-TOPO™ with a pRSET vector. Our pET vectors have a pBR322 origin and our pRSET vectors have a pUC origin, so they are able to replicate in E. coli at the same time. The only issue here is that pRSET is a high copy number vector and our pET vectors are not. Therefore, you may get significantly more protein expression from pRSET in comparison to pET if they are expressed in the same host.

Answer Id: E4181

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Product FAQ

I am trying to express my protein using a T7 promoter-based vector. What does “leaky expression” mean?

Answer

Leaky expression means there is some basal level expression seen. For example, in all BL21 (DE3) cell lines, there is always some basal level expression of T7 RNA polymerase. This “leaky expression” could lead to reduced growth rates, cell death, or plasmid instability if a toxic gene is cloned downstream of the T7 promoter.

Answer Id: E9722

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