Q: How should I store my competent E. coli?

We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

Question 1

Can I store my competent E. coli in liquid nitrogen?

Accepted Answer

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

Question 2

How should I store my competent E. coli?

Accepted Answer

We recommend storing our competent E. coli strains at -80&#176;C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

Question 3

I accidentally stored my E. coli slyD-Extract, E. coli Reaction Buffer (-A.A.), and 2X feed buffer at room temperature. Can I still use them?

Accepted Answer

Unfortunately, this may result in a loss of activity.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 4

I've run out of the T7 RNA polymerase for my cell-free expression. What do you suggest I use?

Accepted Answer

We would recommend using T7 RNA polymerase (Cat. No. 18033019, 50 U/µL). Use 1-1.5 µL in a 50 µL reaction system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 5

I'm getting smearing after running my cell-free expression reaction on a gel.. What could be the cause of this?

Accepted Answer

Smearing may occur if samples for the following reasons:

- Samples were not precipitated with acetone: precipitate proteins with acetone to remove background smearing.
- Too much protein was loaded: reduce the amount used.
- The gel itself was not clean: rinse the gel briefly before exposing to film.
- Ethanol was present in the protein synthesis reaction: make sure that any residual ethanol is removed during DNA purification.
- Check the date of your pre-cast gels: do not use gels after the expiration date.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 6

I'm seeing a ladder of small-sized products after running my reaction on a gel when using the Expressway system. Why is this?

Accepted Answer

There may be several reasons for why this is occurring. The most common are: proteolysis, degradation of DNA and/or RNA templates (truncated templates will generate truncated protein products), internal initiation (if there are many methionines and internal RBS-like sequences in the gene, the ribosome may initiate translation from the wrong methionine), premature termination, translational pausing, frequent rare codon usage, complicated secondary structure of RNA, and others. This can also happen if proteins are denatured for too long, or not enough SDS was added to the 1X SDS-PAGE sample buffer.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 7

With a cell-free expression system, I'm getting good protein yield, but it has low biological activity. What can I do?

Accepted Answer

- Your protein may not be folding properly: try to reduce the incubation temperature to as low as 25 degrees C during synthesis.
- You may require post-translational modification of your protein: the Expressway system will not introduce post-translational modifications to the recombinant protein.
- Your synthetic protein may require co-factors for complete activity: try adding required co-factors to the protein synthesis reaction.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 8

I am using using a cell-free expression system and my control reaction is not working. What should I do?

Accepted Answer

If you are getting no protein from your control reaction, the reagents may have lost activity or may be contaminated with RNases. Check the storage conditions and expiration of the reagents. Use care when freezing and thawing the Expressway E. coli slyD-Extract, Expressway 2.5X IVPS E. coli Reaction Buffer, and Expressway 2X IVPS Feed Buffer. One or two freeze/thaw cycles are acceptable, but avoid multiple cycles.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 9

I am using a cell-free expression system and I'm getting low protein yield with my large protein. Is there anything I can do to increase my yield?

Accepted Answer

Protein yield may decrease as the size of the protein increases. You can try to reduce the incubation temperature to 25-30 degrees C during protein synthesis.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 10

I am using a cell-free expression system and my protein is forming aggregates. What should I do?

Accepted Answer

You can try to reduce the incubation temperature to 25-30 degrees C during protein synthesis. Additionally, a mild detergent can be added (e.g., up to 0.05% Triton-X-100, 0.025% sodium dodecyl maltoside, 0.1% CHAPS, or 0.05% Brij-58) to the reaction and feed buffer. You can also try to add molecular chaperones to the reaction.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 11

I'm trying to express my protein in vitro and am getting low to no protein yield, whereas my control reaction is producing protein. What should I do?

Accepted Answer

Please review the following suggestions:

- Check the sequence of your vector (ATG initiation codon, in frame, etc.).
- If working with a N- or C-terminal tag, the tag may be affecting the RNA structure and lowering translation levels. Try moving the fusion tag or the other terminus.
- Ensure that your DNA template is pure, and not contaminated with ethanol, sodium salt, ammonium acetate, or RNases.
- Do not purify your DNA from an agarose gel, as this can inhibit the reaction.
- We recommend using 10-15 µg of template DNA in a 2 mL protein synthesis reaction. If you are expressing a large protein, increase the amount of DNA template used in the protein synthesis reaction to 20 µg.
- Ensure that you are using a thermomixer or incubator with shaking, as opposed to a non-shaking incubator or water bath for the reaction.
- Multiple feeding steps can further improve the protein yield. Instead of doing one feeding at 30 min of the initial reaction, you can feed the reaction multiple times with smaller volumes of feed buffer to the sample more frequently (i.e., 0.25 mL feed buffer to 1 mL sample every 45 min over 3 hours) after initiating protein synthesis.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 12

How can I analyze my samples after the protein synthesis reaction when using the Expressway expression system?

Accepted Answer

There are several ways to analyze your samples after the protein synthesis reaction, including: Coomassie-stained protein gel analysis, western blot analysis, enzymatic activity assay, or by affinity purification (if an affinity tag is present). If you plan to analyze your sample using polyacrylamide gel electrophoresis, you should first precipitate the proteins with acetone to remove background smearing. A protocol to perform acetone precipitation and other general guidelines for gel electrophoresis are provided in the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) on page 22.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 13

I am using the Expressway system and I'm worried about protein degradation. Can I use a protease inhibitor?

Accepted Answer

If needed, we recommend the addition of PMSF (final concentration 0.5-1.0 mM) at the beginning of the Expressway reaction. It is better to dissolve the PMSF in isopropanol instead of ethanol, as ethanol has a negative effect on protein synthesis. You can also use Pefabloc SC (final concentration 0.1-0.2 mM AEBSF) in your transcription/translation reaction. Both are serine protease inhibitors.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 14

I want my proteins to be glycosylated. Can I use the Expressway system?

Accepted Answer

No, unfortunately, the machinery for glycosylation is absent in these extracts.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 15

Since so many amino acids will code for Arg, will this make it difficult to express GC-rich proteins using the Expressway system? Will the addition of extra Arg to the reaction be helpful?

Accepted Answer

We have not specifically tested for this, although we do know that the limiting factor will be the tRNAs. Because of this, simply adding more of a particular amino acid will not make a difference.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 16

Can I purify proteins produced from the Expressway system?

Accepted Answer

We have not actually done any purifications with the extracts using the His tag. However, it should work, especially if you do it under denaturing conditions.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 17

Will proteins made in the Expressway system form disulfide bonds?

Accepted Answer

No. Disulfide bridges will not form in this system. However, the formation of disulfide bridges may be achieved through the addition of iodoacetamide (Biotechnol Bioeng 2004, 86(2):188-195). Pre-incubation of the extract with 3 mM iodoacetamide for 30 min at room temperature is recommended. The reaction already contains 1 mM DTT (equivalent to 2 mM sulfhydryls); therefore, only 1 mM iodoacetamide will be in excess.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 18

Are there any chaperone proteins added to the Expressway system extracts for protein folding?

Accepted Answer

E. coli cells do indeed contain some chaperone proteins used for protein folding. However, extra chaperone proteins were not added to the extract.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 19

Are there other unnatural amino acids that I can use in my Expressway reaction in place of methionine?

Accepted Answer

Methionine is supplied separately in the kit to allow you to incorporate unnatural amino acids into your recombinant protein and adjust the amino acid concentration in the protein synthesis reaction. Depending on your application, you may use the following unnatural amino acids:

- Radiolabeled methionine: Use ³⁵S-methionine to produce radiolabeled protein for use in expression and purification studies. See “Performing the Protein Synthesis Reaction” on page 21 of the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) for recommended amounts of labeled and unlabeled methionine.

- Heavy metal-labeled methionine: Use selenomethionine (Budisa et al., 1995 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374119/]; Doublie, 1997 [http://www.ncbi.nlm.nih.gov/pubmed/9048379]; Hendrickson et al., 1990 [http://www.ncbi.nlm.nih.gov/pubmed/2184035?dopt=Abstract]) to produce labeled protein for use in X-ray crystallographic studies. See “Performing the Protein Synthesis Reaction” on page 21 of the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) for recommended amounts of labeled methionine. Note: When using selenomethionine, do not use any unlabeled methionine in the protein synthesis reaction.

When setting up the protein synthesis reaction:

- To generate radiolabeled protein using ³⁵S-methionine, use 2 µL of ³⁵S-methionine and 1 µL of unlabeled 75 mM methionine.

- To generate labeled protein using selenomethionine, use 2 µL of selenomethionine only; do not add unlabeled methionine.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 20

What can I do to maximize my protein yield when using the Expressway system?

Accepted Answer

You may obtain your protein of interest in as little as 2.5 hours of incubation after feeding (3 hours total). Many reactions yield 80-90% of total protein within 3 hours. However, for maximum yield, we recommend incubating the reaction for a full 6 hours.

Additionally, higher protein yields may be obtained by adding one half-volume of feed buffer at 30 minutes and one half-volume of feed buffer again at 2 hours after initiating the protein synthesis reaction.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 21

What temperature should I use for the protein synthesis reaction when using the Expressway system?

Accepted Answer

We recommend incubating the protein synthesis reaction at a temperature range from 30-37 degrees C. The optimal temperature to use depends on the solubility of your recombinant protein, and should be determined empirically. Higher protein yields are generally obtained with incubation at higher temperatures (i.e., 37 degrees C); however, protein solubility generally improves with incubation at lower temperatures (i.e., 30 degrees C).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 22

I only have a stationary incubator. Can I use this for my Expressway system experiments?

Accepted Answer

To obtain optimal protein yield, it is critical to mix the reaction thoroughly throughout the incubation period. We recommend using a thermomixer incubator set to 1,200 rpm or a shaking incubator set to 300 rpm. Do not use stationary incubators such as incubator ovens or water baths, as protein yields may be reduced by up to 30-50%.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 23

Can the reaction volume be scaled up or down using the Expressway system?

Accepted Answer

For screening reactions, the standard volume is 100 µL (50 µL initial reaction + 50 µL feed buffer), but this can be decreased to 25 µL reaction volume and increased up to 2 mL reaction volume. Note that protein yields may vary depending on the nature of the protein expressed and the template used.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 24

Can the reaction time go beyond 2 hours for protein synthesis in the Expressway system?

Accepted Answer

The standard reaction time is 2 hours. However, increasing the time to 4 hours may increase the yield of protein. For less soluble proteins, this longer incubation should be carried out at ambient temperature.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 25

Can the proteins made in the Expressway system be glycosylated?

Accepted Answer

No, the proper machinery for glycosylation is not present in these extracts.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 26

Do the Invitrogen pET vectors work in the Expressway system? If not, what vectors do you suggest?

Accepted Answer

Some Invitrogen pET vectors work well in this system; however, the yields might be lower than that found with other vectors due to the presence of the T7lac promoter. The lac repressor can bind to the lac operator site and interfere with expression even when IPTG has been added to the reaction. The best vectors to choose are the pEXP-DEST vectors, pEXP5-TOPO vectors, and pRSET vectors. In addition to the T7 promoter, these also have a gene sequence that enhances translatability.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 27

Is the Expressway S30 extract prepared from a supF or supE E. coli strain? Can the Expressway system be used with modified amino acids?

Accepted Answer

No, the E. coli strain is not a supF or supE strain, and it has very little suppressor activity. Therefore, it should be useful for introducing modified amino acids.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 28

Should a high copy number plasmid be used with the Expressway system?

Accepted Answer

Yes. We recommend starting out with a high copy number plasmid. This way, a researcher can go right from a miniprep kit directly into the Expressway reaction. Many of the pET vectors are low copy, and need to be concentrated before being used in an in vitro expression system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 29

Does the Expressway system help to increase the solubility of proteins?

Accepted Answer

We have looked at two proteins: one that was totally insoluble in intact E. coli and another that was partly insoluble in E. coli. In both cases, we saw at least some soluble protein when synthesized in vitro with the Expressway system. In these instances, it did seem that there is at least some increased solubility with the Expressway system. However, synthesizing protein in vitro does not completely change protein solubility.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 30

Should I gel purify my DNA template before using it in my protein synthesis reaction using the Expressway system?

Accepted Answer

No, we do not recommend doing so, as we have seen this inhibit the protein synthesis reaction. Instead, you can use commercial DNA purification kits (such as our PureLink HQ Mini Plasmid Purification Kit) or a CsCl gradient centrifugation to purify your DNA template.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 31

What is the difference between the pEXP5-NT/TOPO and pEXP5-CT/TOPO vectors?

Accepted Answer

Both vectors contain a T7 promoter, RBS, and T7 terminator with spacing and sequence configuration optimized to allow for high levels of protein expression in the Expressway system. The pEXP5-NT/TOPO vector contains an N-terminal peptide containing the 6xHis tag and a TEV recognition site to allow production of a recombinant fusion protein that may be easily detected and purified. The pEXP5-CT/TOPO vector contains a C-terminal tag containing the 6x His tag to allow for production of a recombinant fusion protein that may be easily detected and purified. Protein yields can vary significantly depending on whether the recombinant protein of interest is expressed as an N- or C-terminal fusion, and therefore, both constructs should be tested.

Question 32

What template should I use for expression using the Expressway system?

Accepted Answer

You can use supercoiled plasmid DNA, linear DNA, or a PCR product as your template. For proper expression, all templates must contain a T7 promoter, an initiation codon, and a prokaryotic Shine-Dalgarno ribosome-binding site (RBS) upstream of the gene of interest. If you are designing your own expression construct, we recommend generating a DNA template with the following elements:

- Gene of interest placed downstream of a T7 promoter and a ribosome-binding site (RBS). The gene of interest must contain an ATG initiation codon and a stop codon.

- Sequence upstream of the T7 promoter containing a minimum of 6-10 nucleotides (nt) for efficient promoter binding (required for linear PCR products). This sequence need not be specific.

- Sequence following the T7 promoter containing a minimum of 15-20 nt, which forms a potential stem-and-loop structure as described by Studier et al., 1990.

- Sequence of 7-9 nt between the RBS and the ATG initiation codon for optimal translation efficiency of the protein of interest. This sequence need not be specific.

- A T7 terminator located 4-100 nt downstream of the gene of interest for efficient transcription termination and message stability.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 33

Are there considerations I need to take into account for my vector design when using the Expressway system?

Accepted Answer

- Transcription of the gene of interest must be driven by a T7 promoter (not the T7lac promoter). Using a T7lac promoter typically renders poor yield, as the lac repressor encoded by the lacI gene binds and represses transcription from this promoter.

- The T7 terminator is important for efficient in vitro transcription from a supercoiled plasmid. If the terminator is absent, long nonspecific RNA products will be produced, which can deprive the reaction of dNTPs and generate a copious amount of pyrophosphate.

- A gene10 sequence enhances the stability of the in vitro expressed sequence. This sequence causes a specific stem-loop structure to form, which helps to stabilize the mRNA and leads to increased translation.

- The mini cistron (in the Trc vectors) also acts to enhance translation by coding for a short gene sequence that encodes a small peptide. Since this brings the translation machinery to the proximity of the start of the gene of interest, it helps to initiate the system downstream.

- We recommend starting with a high copy number plasmid. This way, minipreps can be used directly with the Expressway system.

- Spacing between the RBS and ATG is very important for efficient translation.

- The RBS will increase the yield of protein and increase translation fidelity.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 34

What lysate systems can be used for in vitro protein expression?

Accepted Answer

E. coli, rabbit reticulocyte lysate (RRL), and HeLa cell lysate can all be used for in vitro translation. Our Expressway system utilizes E. coli. In general, RRL efficiently translates proteins greater than 30 kDa. The 1-Step Human In Vitro Protein Expression Kits are ideal for expressing human proteins.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 35

Why is the E. coli extract used in the Expressway system labeled as "sly D"?

Accepted Answer

slyD is an endogenous gene product from E. coli. slyD is very Cys-rich, which makes it interact with the Lumio detection agent.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 36

What is the difference between the Expressway Cell-Free and Expressway Lumio Cell-Free E. coli Expression Systems?

Accepted Answer

The Expressway Lumio system incorporates the benefits of the Expressway cell-free system and Lumio technology. Using the Lumio kit, your gene of interest is fused to a Lumio tag, enabling sensitive and specific in-gel detection of the Lumio -tagged fusion protein in polyacrylamide gels without the need for staining or western blotting. You can also monitor real-time synthesis of the Lumio -tagged protein using a standard fluorometer.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 37

What are some disadvantages of an in vivo expression system that can be overcome with the Expressway Cell-Free E. coli Expression Kit?

Accepted Answer

- Toxicity to the host cell from over-expressed product
- Product insolubility and formation of inclusion bodies
- Rapid proteolytic degradation of the expressed protein
- Incorporation of unnatural or modified amino acids
- Incorporation of fluorescent probes into the protein
- Requirement of high-throughput analysis of protein products

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 38

What are the advantages of using a cell-free expression system?

Accepted Answer

A cell-free expression system is best used when working with a toxic target, as no cells are needed for protein expression. In vitro protein expression utilizes the necessary cellular components to drive expression in a single tube.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 39

Can you give me an overview of the Expressway system?

Accepted Answer

- Begin by generating a DNA template, either by PCR or in a plasmid vector
- Purify the template
- Perform the synthesis reaction
- Analyze the sample via Coomassie staining, western blot, etc.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 40

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

Accepted Answer

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Question 41

Does Platinum Taq DNA Polymerase High Fidelity enzyme mix leave 3' A-overhangs on the PCR product for subsequent cloning into a TOPO TA or original TA vector?

Accepted Answer

Yes, the enzyme mix leaves 3' A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3' A-overhangs to the product for TA cloning.

Question 42

I'm seeing a lot of vector-only colonies when I try to perform a negative control reaction using vector only (no insert) for a TOPO reaction. Is my TOPO vector re-ligating?

Accepted Answer

Using the vector only for transformation is not a recommended negative control.  The process of TOPO-adaptation is not a 100% process, therefore, there will be &#147;vector only&#148; present in your mix, and colonies will be obtained.

Question 43

I'm trying to clone in my phosphorylated PCR product into a TOPO vector, and I'm getting no colonies.  However, when I clone the same product into a TA vector, everything works perfectly.  Why is this?

Accepted Answer

Phosphorylated products can be TA cloned but not TOPO cloned.  This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate.  Phosphorylated products should be phosphatased (CIP) before TOPO cloning.

Question 44

I'm able to get a lot of colonies, however, none contain my insert of interest.  What should I do?

Accepted Answer

You may be cloning in an artifact.  TA and TOPO Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)

Question 45

A majority of colonies are blue or light blue, with very few white colonies.  What should I do?

Accepted Answer

There could be a few possibilities for this:

- The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
- A polymerase that does not add 3' A-overhangs was used. If you used a proofreading enzyme, you will need to do a post-reaction treatment with Taq polymerase to add the 3' A-overhangs.
- PCR products were gel-purified before ligation. Gel purification can remove the single 3' A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
- The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
- Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 µl of the PCR mixture in the ligation reaction.
- The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
On a typical plate there are a few white colonies which do not contain insert. These are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.

Question 46

I'm getting no colonies after transformation.  What should I do?

Accepted Answer

No colonies may occur due to the following problems:

Bacteria were not competent. Use the pUC18 vector included with the One Shot module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 µg/mL of ampicillin or 50 µg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO cloning only). Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. The TOPO vector has a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. The non- TOPO vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.

Question 47

I'm getting low cloning efficiency with my TOPO cloning reactions.  What should I do?

Accepted Answer

Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3' A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3' ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3' A next to another A. Taq is most efficient at adding a nontemplate 3' A next to a C. You may have to redesign your primers so that they contain a 5' G instead of a 5´ T.

Question 48

I'm getting very few colonies after transformation of my TOPO cloning reaction.  How can I increase the number of primary colonies?

Accepted Answer

Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
- Add 100 µL double diH2O to the 6 µL TOPO reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 µL 3 M Na-Acetate, 2 µL 20 µg/µL glycogen, 300 µL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 µL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
- Resuspend pellet in 10 µL ddH2O and electroporate 3.3 µL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 µL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.

Question 49

I'm planning on cloning a 1kb fragment for sequencing and want to minimize the amount of vector sequence in my data.  Which of your vectors should I use?

Accepted Answer

We would suggest using our TOPO TA cloning kit for sequencing, which contains the pCR 4 TOPO vector, or our Zero Blunt TOPO PCR cloning kit for sequencing, which contains the pCR4Blunt-TOPO vector.

Question 50

I'm trying to decide between your pCR2.1 TOPO and pCR4-TOPO vectors to clone a 150 bp PCR product for sequencing.  Which would you recommend?

Accepted Answer

Due to the small size of your product, we recommend using the pCR 2.1 TOPO vector for your cloning.  This size fragment would not be able to fully interrupt the ccdB gene in the pCR4-TOPO vector, and therefore, you may not get colonies as ccdB is lethal to E. coli.

Question 51

What are the insert size limitations of TOPO cloning kits?

Accepted Answer

Regular TOPO TA Cloning kits are efficient for cloning PCR products up to approximately 2-3 kb. With PCR products larger than 3 kb, the efficiency of cloning drops significantly. The TOPO XL PCR Cloning Kit has been optimized for TOPO cloning of long (3-10 kb) PCR products.

If using the regular TOPO kits, here are some tips to improve efficiency:

1. Use crystal violet instead of ethidium bromide (EtBr) to visualize the PCR for gel isolation to avoid DNA nicks
2. Increase incubation time of the TOPO reaction to 30 mins
3. Keep insert:vector molar ratio low, optimally 1:1
4. Dilute reaction to 20 µL, while maintaining same amount of vector and insert. Increase the volume of the salt solution to 3.7 µL to compensate for the increase in volume. Diluting the reaction reduces the competition for the vector ends.

Question 52

Can I store my TOPO vector plus insert reaction?  At what temperature?

Accepted Answer

Storage of the TOPO vector plus insert reaction for 1 week at 4 degrees C has shown no detectable decrease in the cloning efficiency of the TOPO reaction, as >95% of the colonies have insert. However, the total number of colonies was decreased by 10-fold.  Storage of the TOPO reaction mix overnight at 4 degrees C showed little to no decrease in the number of colonies when compared to fresh TOPO reaction mix.

Question 53

What is the difference between a stop solution and salt solution?  What is its function in the TOPO kit?

Accepted Answer

The composition of the 6X Stop solution is 0.3 M NaCl, 0.06 M MgCl2, and the composition of the 6X Salt solution is 1.2 M NaCl, 0.06 M MgCl2.  Stop solution is only included in the TOPO XL Cloning kit whereas Salt solution is currently included in all of the other TOPO cloning kits.  These solutions prevent free topoisomerase from re-binding and nicking the plasmid, which would reduce the number of colonies from a TOPO reaction.

Question 54

What can inhibit the TOPO cloning reaction?

Accepted Answer

When doing a TOPO cloning reaction, 2 &micro;l of a PCR reaction containing up to 10% DMSO or 1.3 M betaine will not interfere with the TOPO reaction.  Formamide and high levels of glycerol will inhibit the reaction.  These reagents are usually added to the PCR reaction to enhance the yield of the PCR product, e.g., to reduce the effect of secondary structure or assist in amplification of GC-rich sequences.  The effects of tricine or acetamide have not been tested on the TOPO cloning reaction.

Question 55

What considerations should I take into account when designing primers for PCR of an insert which will be cloned into a TOPO vector?

Accepted Answer

PCR primers should not have 5'-phosphates when cloning into any TOPO vector, as the presence of 5'-phosphates inhibit the TOPO cloning reaction.  Phosphorylated products can be TA-cloned but not TOPO-cloned.  This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector.  TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate.  Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate.  Phosphorylated products should be treated with phosphatase (CIAP) prior to TOPO-cloning. Treatment with CIAP may raise efficiency to 25%.  PCR products generated with 5'-biotinylated primers (or any other 5'-label including 5'-Cy5) will not ligate into any of the TOPO vectors due to steric hindrance.

Question 56

Do I need to gel purify my PCR product for TOPO cloning?

Accepted Answer

Gel purification is not required if the gel indicates that the PCR product is clean with no visible non-specific bands or primer dimers.  It is recommended if the PCR product is >1.5 kb or if non-specific bands and primer dimers are visible on the gel.  Smaller products clone much more efficiently into the vector than larger products; therefore, they should be eliminated from the sample prior to cloning.  There is some reduction in A-overhangs if the PCR product is gel purified, which along with PCR product loss during the procedure may slightly reduce total number of colonies.  However, the percentage of colonies with insert does not change; it is typically >90% recombinant clones.

Question 57

I typically store my PCR products before TOPO cloning.  Is this okay?

Accepted Answer

For optimal TOPO cloning, we recommend using fresh PCR products.

Question 58

What are the advantages of using a TOPO TA cloning system compared to traditional TA cloning?

Accepted Answer

TA cloning ligates the insert and vector using a T4 DNA ligase, while TOPO TA cloning uses the intrinsic properties of topoisomerase, which ligates the insert and vector during a 5 minute desktop reaction.  TOPO TA cloning results in >95% recombinants, while TA cloning results in >80% recombinants.

Question 59

How do I adapt my cloning vector for TOPO cloning?

Accepted Answer

We offer a custom service for TOPO cloning adaptation services.  Our scientists can prepare your vector for either blunt TOPO cloning, TOPO TA cloning, or directional TOPO cloning of PCR products.

Question 60

Can I order my TOPO vector as a standalone product?  I have plenty of competent cells.

Accepted Answer

Yes, our pCR.1 TOPO TA (Cat. No. 450641), pCR4-TOPO TA (Cat. No. 450030), pCRBluntII-TOPO (Cat. No. 450245) are available separately.

Question 61

Can I run the TOPO vector on a gel?

Accepted Answer

No, we do not recommend this as these vectors contain the topoisomerase DNA protein complex conjugated to the end of the vector.

Question 62

What range of PCR product (molar ratios and ng quantities) do you suggest for TOPO TA cloning?

Accepted Answer

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1 (insert:vector).  The ng quantities should be between 5-10 ng of a 2 kb PCR product in a TOPO cloning reaction.

Question 63

What are some of the prerequisites for TOPO cloning?

Accepted Answer

Please consider the following before TOPO cloning:

- TOPO cloning cannot ligate DNA with a 5' phosphate group.
- TOPO cloning will decrease in efficiency inversely with the size of the insert (above 3 kb) unless using the TOPO XL cloning kit.
- TOPO vectors contain different antibiotic resistance markers which should be considered before purchase.
- TOPO TA vectors accept fragments containing a 3' A overhang while Zero Blunt vectors accept fragments that are blunt-ended.

Question 64

I received my TOPO vector and the solution is colored.  Is it okay to use?

Accepted Answer

TOPO and TOPO TA vectors (non-directional) have phenol red dye added.  The color should be pink (or yellow) at room temperature.  If it turns blue when PCR product is added, the PCR product buffer is too basic and the number of transformed colonies will drop. When the solution is yellow, it signifies an acidic pH.  At a pH 2.0, TOPO vectors still maintain high cloning efficiency.  Directional TOPO and Zero Blunt TOPO vectors have bromophenol blue dye added.

pEXP-5-CT/TOPO™ TA Expression Kit - FAQs

Can I store my competent E. coli in liquid nitrogen?

How should I store my competent E. coli?

I accidentally stored my E. coli slyD-Extract, E. coli Reaction Buffer (-A.A.), and 2X feed buffer at room temperature. Can I still use them?

I've run out of the T7 RNA polymerase for my cell-free expression. What do you suggest I use?

I'm getting smearing after running my cell-free expression reaction on a gel.. What could be the cause of this?

I'm seeing a ladder of small-sized products after running my reaction on a gel when using the Expressway system. Why is this?

With a cell-free expression system, I'm getting good protein yield, but it has low biological activity. What can I do?

I am using using a cell-free expression system and my control reaction is not working. What should I do?

I am using a cell-free expression system and I'm getting low protein yield with my large protein. Is there anything I can do to increase my yield?

I am using a cell-free expression system and my protein is forming aggregates. What should I do?

I'm trying to express my protein in vitro and am getting low to no protein yield, whereas my control reaction is producing protein. What should I do?

How can I analyze my samples after the protein synthesis reaction when using the Expressway expression system?

I am using the Expressway system and I'm worried about protein degradation. Can I use a protease inhibitor?

I want my proteins to be glycosylated. Can I use the Expressway system?

Since so many amino acids will code for Arg, will this make it difficult to express GC-rich proteins using the Expressway system? Will the addition of extra Arg to the reaction be helpful?

Can I purify proteins produced from the Expressway system?

Will proteins made in the Expressway system form disulfide bonds?

Are there any chaperone proteins added to the Expressway system extracts for protein folding?

Are there other unnatural amino acids that I can use in my Expressway reaction in place of methionine?

What can I do to maximize my protein yield when using the Expressway system?

What temperature should I use for the protein synthesis reaction when using the Expressway system?

I only have a stationary incubator. Can I use this for my Expressway system experiments?

Can the reaction volume be scaled up or down using the Expressway system?

Can the reaction time go beyond 2 hours for protein synthesis in the Expressway system?

Can the proteins made in the Expressway system be glycosylated?

Do the Invitrogen pET vectors work in the Expressway system? If not, what vectors do you suggest?

Is the Expressway S30 extract prepared from a supF or supE E. coli strain? Can the Expressway system be used with modified amino acids?

Should a high copy number plasmid be used with the Expressway system?

Does the Expressway system help to increase the solubility of proteins?

Should I gel purify my DNA template before using it in my protein synthesis reaction using the Expressway system?

What is the difference between the pEXP5-NT/TOPO and pEXP5-CT/TOPO vectors?

What template should I use for expression using the Expressway system?

Are there considerations I need to take into account for my vector design when using the Expressway system?

What lysate systems can be used for in vitro protein expression?

Why is the E. coli extract used in the Expressway system labeled as "sly D"?

What is the difference between the Expressway Cell-Free and Expressway Lumio Cell-Free E. coli Expression Systems?

What are some disadvantages of an in vivo expression system that can be overcome with the Expressway Cell-Free E. coli Expression Kit?

What are the advantages of using a cell-free expression system?

Can you give me an overview of the Expressway system?

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

Does Platinum Taq DNA Polymerase High Fidelity enzyme mix leave 3' A-overhangs on the PCR product for subsequent cloning into a TOPO TA or original TA vector?

I'm seeing a lot of vector-only colonies when I try to perform a negative control reaction using vector only (no insert) for a TOPO reaction. Is my TOPO vector re-ligating?

I'm trying to clone in my phosphorylated PCR product into a TOPO vector, and I'm getting no colonies. However, when I clone the same product into a TA vector, everything works perfectly. Why is this?

I'm able to get a lot of colonies, however, none contain my insert of interest. What should I do?

A majority of colonies are blue or light blue, with very few white colonies. What should I do?

I'm getting no colonies after transformation. What should I do?

I'm getting low cloning efficiency with my TOPO cloning reactions. What should I do?

I'm getting very few colonies after transformation of my TOPO cloning reaction. How can I increase the number of primary colonies?

I'm planning on cloning a 1kb fragment for sequencing and want to minimize the amount of vector sequence in my data. Which of your vectors should I use?

I'm trying to decide between your pCR2.1 TOPO and pCR4-TOPO vectors to clone a 150 bp PCR product for sequencing. Which would you recommend?

What are the insert size limitations of TOPO cloning kits?

Can I store my TOPO vector plus insert reaction? At what temperature?

What is the difference between a stop solution and salt solution? What is its function in the TOPO kit?

What can inhibit the TOPO cloning reaction?

What considerations should I take into account when designing primers for PCR of an insert which will be cloned into a TOPO vector?

Do I need to gel purify my PCR product for TOPO cloning?

I typically store my PCR products before TOPO cloning. Is this okay?

What are the advantages of using a TOPO TA cloning system compared to traditional TA cloning?

How do I adapt my cloning vector for TOPO cloning?

Can I order my TOPO vector as a standalone product? I have plenty of competent cells.

Can I run the TOPO vector on a gel?

What range of PCR product (molar ratios and ng quantities) do you suggest for TOPO TA cloning?

What are some of the prerequisites for TOPO cloning?

I received my TOPO vector and the solution is colored. Is it okay to use?

I have a TOPO TA Cloning kit with TOP10 cells. I ran out of competent cells but still have vector left. I also have subcloning DH5? cells and TOP10F' cells in the freezer. Are either of these cells compatible? What strain features should I be aware of?

I'm getting overgrowth of colonies. Why?

I'm only getting white colonies, but none of the clones have an insert. What can I do?

I'm trying to transform large plasmid, 40 kb in size. What strain should I use?

I'm trying to clone a gene that has multiple repeated sequences into my pCR2.1-TOPO vector, followed by transformation into TOP10 cells. My clones contain random rearrangements and deletions. What can I do?

I'm getting no colonies at all on my plates. Can you help?

I'm transforming pCR2.1-TOPO clones into TOP10F' cells. Will I need to add IPTG to my plates along with X-gal for blue/white screening? What if I used TOP10 cells instead?

I'm plating my untransformed TOP10 cells on ampicillin as a negative control, but see a lot of colonies on the plates.

I'm subcloning fragments of yeast genomic DNA into a TOPO vector. I'm seeing a lot of deletions in the clones I'm selecting. What can I do?

I've cloned my gene into the pCR2.1-TOPO vector and transformed into the TOP10 cells that came with the kit. I then did a plasmid miniprep followed by digestion of the DNA with XbaI. However, the vector is not cutting correctly. What happened?

What suggestions can you make for blue/white screening?

I want to store my transformed cells long term. Do you have a protocol for this?

Can I transform 2 plasmids into the same cell?

What concentrations do you typically recommend for X-gal and IPTG for blue/white screening?

How is competent cell efficiency measured? How is it calculated?