Frequently Asked Questions

What are the advantages of gene synthesis compared with traditional PCR cloning?

Compared with traditional PCR cloning method, gene synthesis is accurate and target products without affecting the subsequent verification of gene function.

Codon optimization can improve the expression and solubility of target protein.

The construction of tissue-specific cDNA library by common PCR cloning method is time-consuming and costly.

The gene synthesis turnaround time is fast and cost effective without relying on the template and enzyme restriction site.

Difficult gene sequences that cannot be obtained by traditional PCR cloning techniques but can be synthesized by gene synthesis.

What are the advantages of GenCefe gene synthesis service?

Fast turnaround time: will be shipped starting from 8 days.

High quality delivery: 100% sequence accuracy guaranteed with sanger sequencing method.

Complex & difficult genes synthesis: expertise in synthesizing complex, GC-rich, repeated, or long genes.

Advanced codon optimization technology: GenCefe provides free codon optimization services.

Does GenCefe do extraction of DNA, isolation, cleaning, and purification?

Yes, GenCefe offers plasmid preparation service at different scales and grades, we can also offer customized PCR isolation and cleaning, but not genomic DNA.

Does GenCefe provide services for DNA sequencing, result analysis, and interpretation of kinship between two or more samples?

We can perform DNA sequencing service, we can also analyze/summarize mutations found in the result, but does not provide interpretation of the result.

Can you keep my sequence confidential?

Yes, all customer information will be kept confidential, an NDA (Non-Disclosure Agreement) can be signed with us.

Would it be necessary for codon optimization?

The codon optimization is used for most cases of protein/antibody expression. For example, eukaryotic genes need to be expressed in prokaryotes. Because the codon preference of eukaryotes is very different from that of prokaryotes, so codon optimization of genes can significantly improve the expression efficiency.

Do you have rush gene synthesis service?

Yes. Please contact our project managers to talk about the details of your project.

I received my gene in pUC57, why did you add extra base pair at both end of my sequence?

They are protective bases for blunt end ligation.

What is the storage condition of genes/ constructs you made?

Lyopholized genes can be stored room temperature or -20℃. Once reconstituted, it needs to be stored at 4℃ for short term or -20℃ for long term.

What QC data do I get?

Sequencing raw files, sequencing alignment, COA with restriction digesting analysis result, plasmid map.

What software do I need to open your sequencing result?

The ab1 files are raw files of chromatogram, it can be opened by any sequencing analysis software such as DNAstar, sequencer, or chromas. The SDQ files are alignment which needs to be opened by DNAstar, we also print a PDF file of this alignment which can be opened by Adobe Reader.

Could you purchase some template/ vectors for me from other companies?

Yes, we can try, but some companies will not sell vectors to a commercial entity due to license issue, if you are from academia, it is better for you to purchase it and ship it to us.

In which form do you request plasmid samples are submitted to your lab?

We prefer dry sample on filter paper.  But will accept DNA dissolved in any buffer if required.

Do you sequence verify the clones you made?

All molecular cloning services includes sequence verification.

If you cannot finish the project (molecular biology project), do I still have to pay?

If we cannot deliver the product, you will not be charged. If we can deliver a partial construct, you will need to pay for the part of the work that is performed.

What is order confirmation? Why do I need to confirm?

We send order summery to customer and will need customer to confirm by replying the email. This is to make sure the order information is correct before project starts. Confirmation is required for molecular biology orders.

How do I change or cancel an order?

If the order is not confirmed you can change or cancel it at anytime, once order is confirmed there will be a charge if you want to change or cancel. You need to contact your project manager to change/cancel orders.

Can I use my own FedEx account for shipping?

Yes, if you have a FedEx account, we can use it for the shipping, but handling fee will be charged.

Protein tag selection

1. How to choose protein tags when constructing plasmids?

Commonly used protein tags include 6xHIS, Flag, GST, c-Myc, eGFP/eCFP/eYFP/mCherryeGFP, HA, SUMO, etc. The appropriate tag should be selected according to the purpose of the experiment.

1) 6xHis

6xHis can be inserted to either the C-terminus or N-terminus of the target protein to facilitate the protein isolation and purification. His tag fusion protein can be purified in the presence of non-ionic surfactants or under denaturing conditions. The former is used to purify highly hydrophobic proteins, and the latter is particularly useful in purifying inclusion body proteins. The vectors contain 6xHis: pET-28a(+), pET-28b, pET-22b(+), pcDNA3.1(+)_myc-hisA, pACYC-duet1, etc.

2) Flag tag

Flag tag is widely used in protein expression, purification, identification, functional research and protein interaction.

The vectors contain Flag tag: pESC-His, pFLAG-CMV2, pENTR4-FLAG and so on.

3) c-Myc

The C-Myc tag is a small tag containing 11 amino acids. These 11 amino acids are expressed as epitopes in different protein frameworks and can still recognize their corresponding antibodies. C-Myc tag has been successfully applied in Western-blot, immunoprecipitation and flow cytometry, to detect the expression of recombinant proteins in target cells.

The vectors contain c-Myc: pCMV-MYC, pcDNA3.1(+)_myc-hisA, pCMV-RFP-C-Myc, pCMV-Myc and so on.

4) eGFP/eCFP/eYFP/mCherryeGFP

These tags are enhanced green fluorescent protein, enhanced yellow-green fluorescent protein, enhanced yellow-green fluorescent protein, and monomer red fluorescent protein, which have different excitation wavelengths and emission wavelengths. These protein tags are derived from wild-type fluorescent protein through amino acid mutation and codon optimization.

The vectors contain eGFP: pEGFP-N1, pEGFP-C1, pcDNA3.1-EGFP, pIRES-EGFP, etc.

5) SUMO

SUMO tag protein is a small molecule ubiquitin-like modified protein. As a fusion tag and molecular chaperone for recombinant protein expression, SUMO can not only increase the expression of the fusion protein, but also has the functions of resisting protease hydrolysis, promoting the correct folding of the target protein, and improving the solubility of the recombinant protein. The SUMO tag is also used together with other tags as a specific enzymatic cleavage site.

The vectors contain SUMO tags: pET-SUMO, pET28a-SUMO, etc.

2. Do I need to consider the molecular weight when choosing a protein tag?

Generally speaking, the larger the protein tag, the greater the impact on the function of the protein, so the macromolecular tag is generally only used for detection or protein purification. Common tags with smaller molecular weights are His-Tag, Flag-Tag, HA-Tag, Myc-Tag, which have only a few amino acids, and there are many commercially available antibodies against these protein tags. Using these tags can save costs and time for preparing monoclonal antibodies. The common protein tags of macromolecules include SUMO, GFP, GST and so on.

3. Do I need to remove the protein tag?

Tag removal is usually performed to facilitate the applications of the target protein. Therefore, when designing and constructing the vector, a protease recognition site can be added between the tag protein and the foreign protein sequences, so that the tag of the target protein can be removed by protease cleavage to obtain the complete target protein. These commonly used protease sites are: HRV 3C protease cleavage site, TEV protease cleavage site, enterokinase cleavage site, SUMO protease cleavage site and so on.

There are some tips for tag removal.

1) The specificity of these protease cleavage sites is relatively high, but the cleavage efficiency is different;

2) Except for SUMO protease, other commonly used proteases will leave several amino acid residues after removing the tag. SUMO protease recognizes the spatial structure of the SUMO tag, and then removes this tag completely without leaving extra amino acid residues;

3) The cleavage efficiency of the protease is also affected by the nature of the amino acid residues near the recognition site. Please refer to the reference for details.

4) Some tags are relatively small and have low immunogenicity, and will not affect further research and application if they are retained. However, some large tags, such as Dsb protein, FkpA protein, GST protein, SUMO tag, etc., still need to be removed.

4. Do I need to add the protein tag to the N-terminus or the C-terminus?

It depends on the characteristics of protein structure and positioning. For example, for proteins that are easily degraded and difficult to express, the protein tag at the 5′ end can be used. This not only ensures the stability of the protein, but also has little impact on the immunogenicity of the foreign protein. This method is suitable for epitope screening. However, if the target protein is a secreted protein, it will encounter the problem that the signal peptide at the 5′ end of the protein will be cleaved off by the enzyme during the secretion of the target protein into the Golgi apparatus. In this way, it is not recommended to add the tag to the 5’end.

5. Will it affect the characteristics of the His tag if it is not terminated immediately after the C-terminal His tag?

The principle of His isolation and purification is that the side chain of histidine residues has a strong attraction to solid nickel. It can be used for immobilized metal chelation chromatography (IMAC) to separate and purify recombinant proteins. If no stop codon is added after the C-terminal His tag, the extra amino acids may affect the spatial conformation of the His tag and affect its adsorption effect.

Reference:

[1] Carson M , Johnson D H , Mcdonald H , et al. His-tag impact on structure. Acta Crystallographica, 2007, 63(3):295-301.

[2] Lichty J J , Malecki J L , Agnew H D , et al. Comparison of affinity tags for protein purification. Protein Expr Purif, 2005, 41(1):0-105.

[3] Kim K I, Baek S H, Chung C H. Versatile protein tag, SUMO: its enzymology and biological function. Journal of Cellular Physiology, 2002, 191(3):257–268.

[4] Porath J , Carlsson J , Olsson I , et al. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature, 1975, 258(5536):598-9. Terpe K. Protein Tags[M]. 2005.

[5] Wilkins M R, Gasteiger E, Tonella L, et al. Protein identification with N and C-terminal sequence tags in proteome projects. Journal of Molecular Biology, 1998, 278(3):599-608.

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