Questions Related to Gene Synthesis

1. Can genes be optimized?

A: KMD Bioscience can provide codon-optimized genes designed to meet your exact research specifications. These sequences are always available for your review before your order is confirmed and are typically included in the project quote.

2. How does codon optimization enhance gene synthesis projects?

A: KMD Bioscience’s codon optimization tool can optimize multiple key parameters to stabilize DNA fragments and improve gene expression efficiency. Parameters include, but are not limited to:

--Codon usage bias

--GC content

--mRNA secondary structure

--Custom desired patterns

--Custom undesired patterns

--Repeat sequences (direct repeats, inverted repeats, and binary repeats)

--Restriction enzyme recognition sites (deletions or insertions)

Please note that KMD Bioscience cannot guarantee the final expression of the protein.

3. What is the difference between subcloning and PCR cloning?

A: Both services involve cloning an insert from an existing template plasmid into a different destination plasmid. For our subcloning service, the insert to be cloned already has restriction sites on both sides for cloning. This service is intended for simple restriction/ligation. The PCR cloning service is provided when an additional PCR step is required prior to cloning. For example, this may be necessary to add the correct restriction sites.

4. Gene Resuspension Protocol

A: For resuspension, we recommend the following method.

-- Briefly centrifuge the tube to ensure the contents settle at the bottom of the tube

-- Resuspend the sample in a volume suitable for your needs.

--For example, 20–50 µL of sterile, high-purity water for concentrations ranging from ~0.25 to ~0.1 µg/µL

--The resuspension solution depends on the downstream application—TE buffer may interfere with the activity of various enzymes

--Vortex briefly, place the DNA tube on ice for 2–10 minutes, then vortex again

Parameters for Commonly Used Dyes in Primer Modification

5. How are oligonucleotides quantified?

A: Oligonucleotides are most commonly quantified by measuring their UV absorbance at 260 nm. A single-stranded oligonucleotide dissolved in neutral aqueous solution at a concentration of 33 µg/ml has an absorbance of approximately 1.0 AU at 260 nm. Optical density (OD) is defined as the absorbance (at 260 nm in a 1-cm cuvette) multiplied by the sample volume (in milliliters), therefore, 1 OD unit is approximately 33 µg of ssDNA.

6. What are synthesis scale and yield?

A: Synthesis scale refers to the initial amount of 3'-terminated starting material in the synthesis medium, typically expressed in nmol, such as 25 nmol, 50 nmol, etc. Yield refers to the total amount of final product recovered after all synthesis and purification steps are complete. Yield is typically measured in OD units, such as 3 OD, 5 OD, etc.

7. Is the amount of oligonucleotide obtained the same as the amount indicated on the scale?

A: No. The synthesis scale ordered by the customer refers to the number of 3’ bases at the start of oligonucleotide synthesis. Since oligonucleotide synthesis (or any chemical reaction) is never 100% efficient, the amount of final product recovered after the synthesis and purification steps (yield) will always be less than the starting amount used for synthesis. Yield depends on the oligonucleotide’s size, coupling efficiency, and base composition. Modifications, secondary structure, and purification services further affect the final yield. KMD Bioscience provides a guaranteed yield based on all these factors, which defines the minimum number of oligonucleotides you will receive. Typically, the material produced from synthesis exceeds the guaranteed minimum; however, if your experiment requires more material than the minimum, we strongly recommend selecting a higher synthesis scale.

    KMD Bioscience is committed to providing comprehensive, one-stop services ranging from gene synthesis to protein expression, designed specifically for the research community and the biopharmaceutical industry. Our service process begins with the precise synthesis of genes. Based on the sequence information or target protein provided by the customer, we perform professional gene design and optimization to ensure efficient protein expression. Subsequently, using advanced vector construction techniques, we stably insert the synthesized gene into a suitable expression vector and select the most appropriate host system (including bacteria, yeast, insect cells, and mammalian cells) for protein production. During the protein expression phase, we utilize the latest biotechnologies and expression optimization strategies to ensure high protein yields and correct protein folding. We then employ various purification techniques—such as affinity chromatography, ion-exchange chromatography, and gel filtration—to highly purify the expressed proteins, while conducting rigorous quality control testing to ensure protein purity and activity. Finally, we also provide functional analysis services for proteins, including enzyme activity assays, binding assays, and immunological activity analyses, to comprehensively support our clients’ research projects and drug development efforts. With our professional technical team and extensive industry experience, KMD Bioscience is your reliable partner, helping to drive your scientific innovation and ensure the success of your biopharmaceutical projects.