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Frequently Asked Questions About Our Products And Services

Gene Synthesis / Molecular Biology

Products & Services

How do you optimise synthetic genes?

Using our proprietary software GENEius, the supplied amino acid or DNA sequences will be optimised and adapted e.g. to the codon usage of any organism. Also repeats or hairpin structures are avoided and the GC content is adjusted.
GENEius is also available as a free online tool for our customers.

What lengths of synthetic DNA can you synthesise?

We can synthesise sequences or complete genes from very short fragments up to several thousand base pairs in length. Please contact us for a quote.

What is the difference between a long oligo and a synthetic gene?

Oligos are, due to chemical reasons, limited in length (max. 120-200 bases) and single stranded. The length of a synthetic gene might extend to several thousand base pairs. Synthetic genes are double stranded and generally cloned.

What is the difference between genes delivered cloned into a vector and delivered as linear PCR product?

Although the sequence of the linear gene will have been analysed and sequence congruence will be 100%, the PCR product represents a mixture of correct and incorrect sequences. Therefore, after subcloning not all clones will show the expected sequence. We recommend analysing 6-12 clones for genes of 300-950 bp.
For cloned genes you receive plasmids with the 100% correct sequence.

Do you offer cloning into a vector of my choice?

Yes. We offer additional subcloning of your gene into any vector, for example expression vectors.

Please send your specific vector1 for subcloning together with the printout of the order confirmation to the following address:

Eurofins Genomics
Gene Synthesis Laboratory
Anzinger Str. 7a
85560 Ebersberg
Germany

Which scales of plasmid preparation do you offer?

We offer the following preparation scales:

  • Mini scale plasmid preparation: 1-10 µg Plasmid-DNA
  • Midi scale plasmid preparation: 15-50 µg Plasmid-DNA
  • Maxi scale plasmid preparation: 100-500 µg Plasmid-DNA
  • Mega scale plasmid preparation: 500 µg-2.5 mg Plasmid-DNA
  • Giga scale plasmid preparation: up to 10 mg Plasmid-DNA

More information about our plasmid preparation service can be found here

What quality standards can I expect?

We verify each synthetic gene via double-stranded DNA sequencing by our in-house sequencing service. We ensure 100% sequence accuracy of every cloned gene.

The high quality standard of the complete synthesis process is permanently assured by our professional quality management. We are certified according to ISO 9001:2000.

What are the turnaround times?

The standard turnaround times (plus one day for the shipment) are:

Standard Genes (up to 1000 bp) 6 working days
Standard Genes < 2000 bp 15 - 20 working days
Standart Genes < 3000 bp 20 - 25 working days
Subcloning additional 5 - 10 working days
Complex Genes additional 5 - 10 working days

The turnaround times for our Express Genes are:

Express Genes (up to 1000 bp) 4 working days
Express Genes (up to 1500 bp) 6 working days
Express Genes (up to 2000 bp) 7 working days
Express Subcloning additional 4 working days

Will my data be treated confidentially?

From the synthesis of the oligos, over the gene synthesis itself up to the quality control by DNA sequencing, Eurofins Genomics offers a complete in-house process, to ensure 100% confidentiality.

How to order

How do I order a synthesised gene?

Standard and complex genes can easily be ordered through our Ecom system.

Alternatively, you can also use the quote request form on our website.
This also applies for Gene Evolution Libraries.

Upon the entry of your offer request, our experts will contact you immediately.

Ordering GeneStrands from FASTA files

The GeneStrands order page provides, additionally to the single entry format the possiblitlity to upload or to copy & paste gene names and sequences. The latter is particularly useful in case of ordering many GeneStrands at once.

If your DNA sequences are available in FASTA format, please use the “Copy&Paste” entry format.

In case you want to copy & paste sequences of, e.g. a FASTA file into the sequence box of the single input page, please ensure that all line breaks in the sequence are removed:

  • Copy your FASTA sequence in an MS Word document
  • Replace the line breaks “^p” (paragraph mark) by a space character (blank)
  • Then you can copy & paste the sequence in your word document into the sequence field on the order page.

Can I track the status of my gene synthesis order?

You can track the order status as well as the delivery status if you have placed your order via our Ecom system. In your personal Ecom account all your order data are stored.

Find your order history via the "my orders" button where you can track the process status of your orders at any time and download your order confirmations and delivery notes of a period of two years.

In case of any questions please contact our Customer Support Team.

Facts & Myths around gene synthesis

It is not possible to synthesise complete plasmids

ItsaMyth

This is a myth. With our gene synthesis protocol we can not only synthesise genes of any length, we can also synthesise complete plasmids.

Synthetic genes are often created by assembling overlapping DNA oligonucleotides. With this method it is possible to create long fragments that will be finally assembled to one large synthetic gene. By adding a circularisation procedure it is therefore possible to even create complete plasmids of any length. As we routinely work with E. coli, your “gene” (i.e. the new plasmid) must harbour an E.coli selection marker and an origin of replication, of course.

Still the question remains, why someone would like to create his / her own plasmid. There are quite a few answers to that, like:

  • Free choice in antibiotic resistance, promoters, origin of replication, etc.
  • Free choice of the multiple cloning site, tailor-made for your projects
  • No potential licensing issues if you want to use your plasmid commercially
  • Sequence optimization of the vector to the host organism, to regulate the expression levels of the gene(s) (e.g. antibiotic resistance etc.)

With SDM I can only mutate one short target region 

ItsaMyth

The fact is:

With our proprietary SDM technology we can efficiently mutate, insert or delete up to 150 bp at the target site within your plasmid. If you only want to target one site, e.g. to test a different amino acid in the active domain of your protein, Standard SDM is the method of choice. And if you have more than just one target site, SDM also is the perfect way to get the multiple-sites-mutated plasmids very fast and cost efficient. With our multi SDM technology we can simultaneously mutate up to 5 sites in just one round. And, of course, also these multiple mutations can be up to 6 bp mutations, insertions or deletions

It is not possible to optimize non-coding sequences

Fact

Due to the redundant nature of the DNA code more than one triplet codon can code for an amino acid e.g. Arginine - CGT, CGC, CGA, CGG, AGA, AGG. The frequency of these codons differs across species. It is therefore possible to optimize a DNA sequence so it contains the same frequency of codons as seen in the organism where the gene will be expressed. The optimization also helps in the reduction of high GC regions and repeat regions in the gene.

This type of optimization is only possible however when the gene to be synthesized (or part of it) is coding for a protein sequence and therefore has codons that can be manipulated in this way. A non-coding stretch of DNA does not contain codons coding for an amino acid and therefore an optimization exploiting the redundancy of DNA cannot be done.

All gene optimisation softwares use only the “best” codons in the optimisation process and a high codon adaption index (CAI) is best

ItsaMyth

The fact is:

Eurofins’ optimisation software GENEius does not only use the best codons. We have shown that this very simple approach does not result in highest protein expression.

During the optimisation process GENEius randomly as­sembles the DNA sequence and then analyses it in relation to codon usage by comparing it to the input codon usage table. This input codon usage table is usually taken from the Kazusa Codon Usage Database (http://www.kazusa.or.jp/codon), but it can also be provided by the customer. GENEius does not simply aim for a high codon adaption index (CAI), instead it harmonises the codons used. Frequently used codons from highly expressed genes are more often used in the resulting gene sequence than less frequently used codons. Very rare codons, however, will be completely avoided. During adap­tion GENEius also checks for “bad motifs” like restriction sites and avoids artificial splice sites, unspecific transcription factor binding sites, etc. Also, to minimise RNA structure direct and in­verted repeats are avoided as they not only make synthesis more difficult, they can decrease DNA stability and reduce efficiency of transcription and translation in E.coli. And last but not least, the GC content is equally distributed to avoid GC-rich subse­quences within in the gene. All these parameters are taken into account and an “optimisation score” is constantly being calculated. If this score falls below a certain threshold, the sequence is taken as the output. This procedure results in a different DNA sequence every time the optimisation is running.

 

If an insert does not harbour restriction enzyme sites from the multiple cloning site (MSC) of your plasmid, the gene can still easily be subcloned into this vector.

Fact

This is a fact, indeed. You only need to cut your vector of choice with one or two suitable enzymes of the MCS. Your gene insert can then be PCR amplified with primers that generate homology to the vector at the ends of the PCR product. These homologous regions can then be used for subcloning via SLIC (= sequence and ligation independent cloning) into your vector. Any gene internal restriction sites do not matter at all because the PCR product won’t be cut during subcloning. Eurofins’ molecular biology experts will do the design and make sure that all your requirements are met, e.g., in frame cloning. SLIC is as fast and efficient as traditional subcloning via restriction enzyme sites and, if you have forgotten, e.g., to introduce a stop codon or a fusion tag at the end of your gene, this can also be fixed simultaneously.

Codon optimisation is the only advantage of gene synthesis

ItsaMyth

The fact is:

Well, yes, most scientists  use gene synthesis for expression improvement of their genes of choice in heterologous systems. This can be achieved via codon usage adaptation using Eurofins’ gene adaption and optimisation software “GENEius”. But there are many more advantages for using gene synthesis. Imagine your NGS data reveal a very interesting DNA sequence that you now want to analyse further. With gene synthesis you can simply order your DNA or RNA sequence, you just need to know the in silico sequence. Another advantage of gene synthesis is fast and reliable access to cDNA sequences that a few years ago would have been done via labour-, time- and cost-intensive cDNA synthesis. No RNA extraction, no RT-PCR, and no RACE is needed for getting perfect full-length cDNA when using gene synthesis. Unwanted restriction sites can be avoided during adaption and subcloning into your own expression vector is also possible. Of course you can combine your gene sequence together with promoter and enhancer elements, polyadenylation signals, restriction sites, etc.

 

Eurofins pEX Standard vectors cannot be used for RNA production or protein expression

Fact

Well, yes, this is a fact (but please see our tip below!). If you only order your open reading frame with restriction sites, this is true. Our pEX vectors are based on pUC18 and do not contain a promoter or terminator for expression of your subcloned gene. They are standard cloning vectors for E.coli. For transcription and translation experiments the gene would have to be subcloned into an expression vector of your choice, harbouring a suitable promoter and potentially a terminator sequence. By the way, for best expression results the ORF should be optimised using our powerful algorithm “GENEius”.

 

And here is a tip for saving time and money:

Include a promoter sequence upstream of your gene sequence. Of course you could also add a terminator sequence at the 3’ end. Then, the pEX vector can be used as an expression vector for protein expression in vivo or, after linearisation, as template for in vitro transcription.

If the standard vector contains my restriction enzymes I will have problems with downstream cloning projects

ItsaMyth

The fact is:

The standard vectors from Eurofins have been designed to have few restriction enzymes in the multiple cloning site (MCS). Even if your restriction enzyme is present in the MCS this will not cause a problem. The extra fragment will be so small that it will not be visible on an agarose gel. All restriction sites in the MCS are at least 4bp (in most cases >10bp) away from your gene so the efficiency of the restriction digest will also not be affected.

In the very unlikely event that one of your chosen restriction sites is also in the vector backbone and a band the same or similar length as your gene is present after the digest, there are a number of possibilities to get around this problem.

Either a 3rd enzyme can be used in the digest that cuts in the vector backbone reducing the size of the unwanted band. In the majority of cases the required band can then be easily gel eluted from an agarose gel.

If this strategy does not work due to the lack of an appropriate restriction site then the gene can be amplified with PCR primers. It is then possible to digest the PCR product with the required enzymes and proceed with the subcloning. It is important in this case to add 2-3 bases to the 5´ ends of the PCR primers as overhangs so the efficiency of the digest is not reduced.

 

 

Technical Questions

In which applications can I use synthetic genes?

Synthetic genes might be used to adapt the codon usage for optimising gene expression, for protein over expression and protein engineering; as standards for Real Time PCR and PCR or to construct hybrid genes or produce DNA vaccines. You can also create multiple variants of a gene.

How should a gene be designed?

The desired gene coding for an artificial protein is divided into a series of 5´-end overlapping complementary oligonucleotides. To achieve maximal benefit from the gene assembly, the sequence of the oligonucleotides to be assembled has to be planned carefully with the following considerations:

  • The formation of hairpins of 4 or more bases should be avoided.
  • Sequences that are commonly associated with poor coupling efficiency during the chemical synthesis should be avoided.
  • It is recommended to avoid sequences that introduce rare codons into a gene, if you want to achieve high levels of gene expression (protein production).
  • The generation of unique restriction sites within the assembled gene will allow subsequent manipulations by recombinant DNA techniques.

Eurofins Genomics uses their proprietary software GENEius for the design of the oligonucleotides.

Are different assembly techniques possible?

Basically, there are three different approaches for the assembly of synthetic genes. In the approach developed by Khorana (Gupta et al., 1968) a series of sequentially overlapping oligonucleotides are synthesised. After the annealing of the oligos, a double stranded DNA fragment containing nicks on both strands is formed. The nicks are sealed in a reaction with DNA ligase, an enzyme that catalysis the formation of phosphodiester bonds between the 5´-phosphate of one double strand oligonucleotide fragment and the 3´-hydroxyl terminus on another adjacent double strand oligonucleotide fragment.

An alternative strategy has been developed, making use of the possibility of synthesising very long oligonucleotide chains. In this approach (Rossi et al., 1982) two oligonucleotides are constructed which upon annealing their 3´-ends overlap. This construct is completed to a full length double strand by a subsequent filling-in reaction using a DNA polymerase. After polymerisation overhanging ends are generated on the double strand fragment by digestion with an appropriate restriction enzyme.

Usually, all methods are followed by the molecular cloning of the gene. Therefore it is necessary to consider the cloning steps while developing the assembly strategy. Larger genes can be divided in sub fragments that are assembled separately. After sequence verification of the cloned sub fragments, they are assembled to the full length construct.

How can the molecular weight of a plasmid be determined?

Every bp has a specific molecular weight, in average each bp ~  0.65 kilodaltons. Therefore, if your plasmid was 3,000 bp long, the approx. molecular weight would be 3,000 bp x 0.65 kDa per bp = 1,950 kDa.  

If you have ordered a synthetic gene subcloned in our standard vector pEX-A2 (2,450 bp) and your gene is 1,000 bp long, the total length of the plasmid would be 3,450 bp and the molceular weight 2,243 kDa.

Is site directed mutagenesis or de novo synthesis preferable?

If mutations are abundant and distributed across the whole gene, we recommend de novo synthesis. De novo synthesis also gives you the opportunity to optimise other features of the gene such as codon usage, GC content, restriction sites etc. Site directed mutagenesis is used if modifications are few, or are clustered in a small part of the gene.

I want to use the genes or GeneStrands in qPCR. Are those products free of cross contaminations?

The synthetic genes and GeneStrands are produced on open deck robots throughout the process, therefore we cannot exclude the possibility that slight cross contamination occurs in rare cases. We have established daily cleaning and decontamination routines to make sure that we deliver the best products possible. If you want to use products from the same order in qPCR / very sensitive PCR reactions, we recommend that you contact us prior to ordering to discuss your specific needs in advance. 

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