What are the methods of purification?

The common purification methods are DSL, OPC, PAGE, HPLC and Dual PAGE & HPLC.

Will the primers degrade when transported at room temperature?

It will not degrade, and the dry primer can be stably stored at room temperature for at least two weeks. The average transport time is usually 5days, so the primers you receive will not degrade.

How many primers of OD value should be synthesized?

According to the purpose of the experiment. In general, about 20 bases of primer 2 OD in PCR amplification can do 400 rxns standard PCR reactions of 50 μL system. If it is to do gene splicing or annealing to do the ligation, 1 OD is enough.

What are the basic principles of primer design?

(1) Primer length generally between 15~30 bases.(2) Primer GC content between 40% and 60%.(3) Bases should be randomly distributed.(4) There should be no complementary sequences in the primers themselves and between the primers(5) The primers should have relatively high ∆G values at the 5’end and middle ∆G values, and low ∆G values at the 3’ end.(6) The single strand of the amplification product should not form a secondary structure.(7) The primers should have specificity.

What causes low fluorescent signal in amplification curves?

First check the baseline-corrected or ROX-corrected raw curves. Low signal values in the amplification curves are mostly due to high background fluorescence. In probe-based detection, high background fluorescence is often caused by poor probe design, leading to insufficient quenching by the quencher group, improper pairing of the reporter and quencher groups, or low fluorescence labeling efficiency of the probe.

What is the principle of oligo synthesis and what types of services are offered?

Oligo synthesis utilizes solid-phase phosphoramidite chemistry. The main types of oligonucleotides we synthesize include:(1) DNA oligos;(2) RNA oligos;(3) Probes;(4) siRNA;(5) sgRNA;(6) miRNA;(7) ASOs;(8) Various modified oligos.

What contaminants are present in the crude product?

Crude oligonucleotides contain the desired product, failure sequences and chemically modified by-products.

Oligo primers in freeze-dried state are highly stable and can be stored at -20°C for at least one year. After reconstitution, dissolved oligos can be stored as a 100 μmol/L stock solution, aliquoted into multiple tubes, and kept at -20°C for at least six months (frequent freeze-thaw cycles may reduce shelf life). Before use, dilute the concentrated solution to the working concentration for your experiments.

How to dissolve oligos?

Each OD of primer needs to be diluted with water to a concentration of 100 μmol/L (equivalent to 100 pmol/μL).You could add an appropriate amount of nuclease-free double-distilled water (pH >6.0) or TE buffer (pH 7.5-8.0) according to your experimental needs.Before opening the vial, centrifuge it at 3000-4000 rpm for 1 minute to prevent the loss of primers when opening.

Why the primers that have been lysed are not good when they were used normally and then used again after a period of time?

If the pH of the water you lysed the primers in is too low or contaminated with bacteria or nucleases, it will degrade the primers. Insufficient thawing and mixing of the liquid when using may also cause inaccurate primer addition. It is recommended to split the primers, avoid repeated freeze-thawing, and use 10 mM Tris pH 7.5 buffer to dissolve the primers. There is also a possibility that the primers are not faulty, but that the quality of the materials used for PCR, especially the template, is not exactly the same as previously used.

Why do long chain primers cost more than short chain primers?

When combining long chain primers, more reagents are required, especially primers longer than 90 base. Due to the increase in cost, the price is higher.

What customization options are available for custom DNA oligos?

The entire process from synthesis, QC to packaging can be highly customized. Customizable options include, but are not limited to:(1) Packaging in 1.5 mL, 15 mL, or 50 mL tubes(2) Capillary electrophoresis analysis(3) High-performance liquid chromatography (HPLC) analysis(4) Polyacrylamide gel electrophoresis (PAGE) analysis(5) Quantitative PCR (qPCR) analysis(6) Full-wavelength scanning(7) OD260/280 measurement(8) Provision of 1 OD sample with each shipment(9) Specialized consumables(10) Customized Certificate of Analysis (COA)

Sometimes the dried primers are yellowish-brown, is this the color of DNA itself?

Synthetic primers may be yellowish brown, white or transparent, which is related to the base composition of the primers and the preparation process of synthesis, primers with high A and G content in the sequence and large OD value are usually yellowish brown, so the yellowish brown primers will not have any effect on the experiment.

How to test the purity of primers?

A common method used in laboratories is the PAGE. For electrophoresis, use a polyacrylamide gel with 7 M urea, 20% polyacrylamide gel for primers with less than 12 bases, 16% polyacrylamide gel for primers with 12-60 bases, and 12% polyacrylamide gel for primers with more than 60 bases. Take 0.2-0.5 OD primer, dissolve with urea saturated solution or add urea dry powder into primer solution until saturated, and heat denaturation (95 ℃, 2 min) before loading sample. The purpose of adding urea is to denaturate, and to increase the specific gravity of the sample, which is easy to add the sample. After a certain period of time (about 2-3 hours), strip the polyacrylamide gel and detect the band type with a fluorescent TLC plate under the UV lamp. There is no impurity under the main band, indicating that the purity is good.(Sometimes due to insufficient denaturation, there may be bands above the main band, which are primer secondary structure bands.)

How to measure the OD value of primers?

The OD value of the synthetic primer was determined by using an ultraviolet spectrophotometer with a wavelength of 260 nm and a quartz colorimetric cup with an optical path of 1 cm to determine the optical density of the solution. The optical density of the solution should be diluted to 0.2-0.8. After the DNA dry powder is fully oscillated and dissolved with a certain volume of water, the OD value is measured by diluting with 1 mL of water. OD value of mother liquor should be converted according to dilution ratio. For example, to verify whether the amount of 2 OD primer is accurate, the simple practice is to add 1 mL water, thoroughly dissolve and mix, take 100 μL, add 900 μL water, a quartz colorimetric cup with a light diameter of 1 cm, a wavelength of 260 nm, and the absorbed reading at this time is 0.2.

When PAGE electrophoresis is performed, why are the swim bands of Oligo DNA of exactly the same length not in the same position?

This situation is more likely to happen when the Oligo DNA is shorter, and the difference between long-stranded Oligo DNA is smaller. There are two main reasons.(1) different components of A, G, C and T and different electrophoresis speeds.(2) The steric structure of DNA is different and the electrophoresis speed is different.

Why does the EB staining vary in shade for the same OD detected by PAGE?

The amount of double-stranded DNA (e.g. plasmid DNA) can usually be judged by EB staining, because EB stains nucleic acids by embedding them between the double helix. Synthetic single-stranded DNA, on the other hand, can only be stained by EB by forming a local hairpin loop structure by folding back on itself or by forming a partial double helix structure between the strands. Due to the different base composition, the possibility of forming secondary structure differs from primer to primer, and the degree of EB staining will also vary, for example, Oligo(dT), etc. does not form secondary structure, and the EB staining effect is very poor. Therefore, EB staining cannot be used for quantification, and UV spectrophotometry should be applied for detection.

What is the longest primer that can be synthesized?

The longer the primer, the higher the probability of problems. Unless there is a special need, we recommend not to synthesize fragments longer than 80 mer. According to the current primer synthesis efficiency, the percentage of full-length primers will not exceed 40% for a crude product of 80 mer, and a lot will be lost in subsequent processing, so the final yield is very low. The maximum length of primers that can be synthesized is 145 bases.

Can I use Agarose gel electrophoresis to analyze the synthetic primers?

It is important to use denaturing PAGE electrophoresis for primers. Since the primers are single-stranded DNA, it is easy to form a complex steric structure, so when Agarose electrophoresis is performed, it is easy to have multiple bands or no bands, and it is even more impossible to quantify by Agarose electrophoresis.

Why is the yield of modified primers lower and the price higher than that of general primers?

(1) Stability and Efficiency: Modified monomers are less stable and have longer coupling times, resulting in lower efficiency and yield.(2) Purification: They require intensive purification methods like PAGE or HPLC, leading to significant material losses.(3) Raw Material Cost: The raw materials for modified primers are much more expensive, often hundreds of times more than those for general primers.

Is there a modification you would recommend to block a ligation reaction at the 3' end of an oligo?

There are several modifications that can block 3' extension. Most 3' modifications will block extension during PCR.The modifications most frequently used for this purpose are the 3'ddC, 3' Inverted dT, 3' C3 spacer, 3' amino, and 3' phosphorylation.

Why is PAGE purification not available with fluorescent dyes?

We do not recommend PAGE purification for dye labeled oligos due to the presence of ammonium persulfate, a component of the PAGE gel. Ammonium persulfate can break linkages and may damage the ring structures that comprise many fluorescent dyes.PAGE gels are exposed to UV light when the oligos are excised from the gel. UV light exposure can also damage fluorescent dyes. HPLC purification provides a pure product without damaging fluorescent modifications.

What causes low fluorescent signal in amplification curves?

First check the baseline-corrected or ROX-corrected raw curves. Low signal values in the amplification curves are mostly due to high background fluorescence. In probe-based detection, high background fluorescence is often caused by poor probe design, leading to insufficient quenching by the quencher group, improper pairing of the reporter and quencher groups, or low fluorescence labeling efficiency of the probe.

What causes erratic peaks in melting curves?

Erratic peaks in melting curves can result from contamination in the reaction system. Confirm contamination using NTC and NRC results, and check potential sources such as water, oligos, enzymes, and the experiment settings. Reagents exposed to strong light or high temperatures may degrade, so it is recommended to compare results with fresh reagents.

Why do dissolved primers work fine at first but not after a period of time?

If the water in which you dissolve the primers has a low pH or is contaminated with bacteria or nucleases, the primers will degrade. Inadequate thawing and mixing during use, and uneven liquid may also cause inaccurate amounts of primers. It is recommended to aliquot primers, avoid repeated freezing and thawing, and use 10 mM Tris pH 7.5 buffer to dissolve primers. There is another possibility that there is no problem with the primers, but that the quality of the materials used in PCR, especially the template, is not completely consistent with that used previously.

Is it related to the primers that PCR amplification does not work?

Basically not. Nowadays, various PCR amplification techniques and high-temperature polymerases have been developed to solve the problems of amplification failure and low amplification efficiency encountered in PCR amplification. For example, slot PCR amplifies gene fragments with very low copy number. Some repeat fragments, fragments with high GC content amplification, must use special amplification means to amplify.Amplification does not come out, mainly the following two situations are more common.(1) RT-PCR. many genes are difficult to amplify by conventional RT-PCR methods. the key to successful RT-PCR lies in the quality of the RNA of the RT reaction and the content of the target gene in the particular tissue and cell.(2) Amplification from the genome. Generally, genes are single copies in the genome, and the genome needs to be used as a template in strictly controlled amounts. Too much genomic DNA can affect the Mg2+ concentration and pH in the reaction system.

PCR amplification has strong non-specific bands, does it indicate that the primers are contaminated?

No. We have analyzed some non-specific bands and sequencing found that at least one primer sequence can be found at both ends of these non-specific fragments. Therefore non-specific amplification is usually the result of template contamination (e.g. contaminated genome in RNA) or unsuitable amplification conditions.

Tsingke ensures sample uniformity by isolating monoclonal clones. Each clone is validated by Sanger sequencing, covering the entire gene and at least 50 bp of the vector backbone upstream and downstream, which ensures the gene is correct and properly placed in the vector. The plasmid then undergoes NGS to verify the accuracy of the entire plasmid and detect any contamination. Before shipment, a strict QC review is conducted.

What are the main types of capture probes?

Cancer research probes, basic research probes, hematologic probes, pathogenic probes, genetic disease probes, methylation probes.

What are the key factors to consider when designing multiplex PCR primers?

1.Primer Length: Primers should be 18-24 bases long to minimize the likelihood of primer dimer formation.2.Tm Values: The melting temperatures (Tm) of all primers in the multiplex reaction should be similar to ensure uniform annealing.3.3'-Nucleotide Complementarity: Avoid complementarity at the 3' ends of primers to prevent the formation of primer dimers.4.Individual Optimization: Optimize each pair of primers independently before pooling them. Once pooled, mix and optimize them sequentially.

Why annealing temperature and cycle number are improtent in multiplex PCR?

The optimal annealing temperature for each primer pair needs to be determined individually, and the lowest Tm value among the primers should be used for efficient annealing in the multiplex reaction. Additionally, using the minimum number of amplification cycles necessary to achieve sufficient product yield helps ensure the efficiency and specificity of the PCR.

Why is it important to optimize reagent concentration and extension time in multiplex PCR?

In multiplex PCR, multiple templates are amplified simultaneously, which can make enzyme and nucleotide concentrations limiting factors. Additionally, the time required to fully synthesize all products increases. Multiplex PCR requires longer extension times compared to single-target PCR to ensure complete synthesis of all products.

How should fluorescent labeled probes be stored?

Multiplex PCR primers should be 18-24 bases long. Longer primers are more likely to form primer dimers.

What happened when sequencing found mutations in the primers?

Primer synthesis is a multi-step chemical reaction, and the efficiency of each step of synthesis is 99% at most, and by-products cannot be avoided. The longer the chain, the higher the cumulative frequency of mutations. When you clone and sequence after PCR amplification, in order to save time and improve the success rate, we have the following suggestions.(1) Please prepare 2~3 positive clones of the bacteriophage after you detect the positive clone, and try to send 2 or more clones for sequencing, so that the success rate will be greatly improved and a lot of time will be saved.(2) You can also send one clone for testing first, and store the rest two clones of the bacteriophage in the refrigerator at 4 degrees, and send the remaining two clones for testing as soon as individual point mutations or deletions occur.(3) This will have a very high probability of getting the correct sequence and will eliminate the need for a series of experimental operations such as re-PCR, ligation, cloning and screening, saving even more time.If you find more than 2~3 clones with mutations in the primer region, and it is confirmed that it is caused by the primer, we will arrange for the expedited free recombination immediately and send it to you as soon as possible.

What is the maximum length and the types of RNA oligos synthesized by Tsingke?

We specialize in synthesizing RNA oligos up to 180 nucleotides in length, and the types mainly include siRNA, sgRNA, miRNA, ASO, and various modified RNAs.

How do you ensure the quality and accuracy of synthetic RNA oligonucleotides?

All RNA oligos undergo rigorous quality control:(1) Mass spectrometry (MS): Every batch is verified by MS to confirm identity and mass accuracy.(2) HPLC purification: Available for >90% purity requirement applications.(3) Nuclease-free and integrity checks: Gel electrophoresis and functional validation are optional upon request.We guarantee sequence accuracy, modification integrity, and high batch-to-batch reproducibility.

What roles do positive and negative controls play in RNAi experiments, and how should I select?

Positive controls are verified siRNAs targeting housekeeping or reporter genes, used to assess the feasibility of the experimental methods. Tsingke provides effective siRNA positive controls targeting GAPDH, ACTB, GFP/EGFP.Negative controls are non-specific siRNAs used to demonstrate the specificity of siRNA effects and can be selected from universal or scrambled sequences based on requirements.

Should the results of negative controls be the same across different groups? What if there is significant deviation?

Normally, the results of negative controls should be similar across different groups. Significant deviations may indicate issues with the accuracy of experimental results.For certain genes, particularly those related to cellular stress or immune response, varying responses to external stress might cause inconsistent gene expression across control groups.

What should I do if the silencing effect is not satisfactory?

The two most common reasons for unsatisfactory silencing effects are low transfection efficiency and suboptimal siRNA sequence design.(1) Check Transfection Efficiency: If you are using siRNA for the first time or working with a new cell line, we recommend testing and optimizing the transfection efficiency. If you have already optimized the transfection conditions and the problem persists, consider switching to a different transfection reagent or method, as this might improve transfection efficiency.(2) Evaluate siRNA Sequence Design: If the transfection efficiency has been improved but the silencing effect is still not satisfactory, it may be due to ineffective siRNA sequence design.

Why is no fluorescence observed after transfection?

Usually, FAM shows green fluorescence (excitation wavelength 495 nm) and CY3 shows red fluorescence (excitation wavelength 550 nm) after transfection. If no fluorescence is observed, consider the following:(1) Is the fluorescence excitation wavelength correct?- Ensure that the excitation wavelengths are set to 495 nm for FAM (green) and 550 nm for CY3 (red).(2) Were the cells washed immediately after transfection?- Wash the cells immediately after transfection to observe fluorescence. Delaying this step can result in reduced fluorescence.(3) Was the siRNA product stored properly?- Improper storage can lead to siRNA degradation and FAM fluorescence quenching. FAM is particularly sensitive to light, so store it away from light.(4) Was the transfection performed away from light?- Operate away from light during transfection. Avoid exposing FAM to white light for extended periods.(5) Was fresh medium replaced in time after transfection?- Prolonged transfection times without timely replacement of fresh medium can affect fluorescence. Ensure fresh medium is added promptly post-transfection.

Why is there a lot of cell death after transfection?

Poor cell health before transfection can lead to increased cell death. Ensure cells are in optimal growth condition, as healthy cells better tolerate transfection reagents. High concentrations of siRNA or transfection reagents can be cytotoxic, so adjusting these concentrations may be necessary. Some transfection reagents are more toxic; consider switching to a less toxic alternative. Low-purity siRNA may contain impurities that affect cell viability; therefore, using high-purity siRNA is recommended. Additionally, prolonged transfection times can exacerbate cell death; ensure timely replacement of fresh medium after transfection.

Why is there low efficiency of cell transfection?

Low efficiency of cell transfection can occur due to several reasons:Insufficient siRNA purity may lead to RNA enzyme contamination. Use DEPC-treated consumables and reagents to prevent this issue.Adjusting the concentration of the siRNA-transfection reagent complex can enhance transfection efficiency.Serum in the transfection mixture can interfere with efficiency. Prepare the mixture without serum to minimize this effect.Avoiding antibiotics in the culture medium is recommended as they can inhibit transfection efficiency.

How long does siRNA work within cells? When is the best time to perform detection?

siRNA-mediated RNA interference (RNAi) is a transient phenomenon and cannot be stably propagated through cell generations. Its effects typically do not last for an extended period, and it is generally recommended to complete detection within 3-4 days post-transfection. The optimal detection time varies depending on the cell type and target gene, but it is usually between 24-48 hours post-transfection. It is generally suggested to assess mRNA levels at 24-48 hours and protein levels at 48-72 hours post-transfection.

Is transfection efficiency related to the siRNA sequence?

Transfection efficiency depends on the characteristics of the cells and the transfection method used, and it is not directly related to the siRNA sequence. Therefore, siRNA transfection efficiency may vary across different cell types.

If the interference effect of the target gene is only about 60%, is the package considered invalid?

Usually, the package products are guaranteed to interfere with about 70% of the target gene, if the interference effect is about 60%, we also consider the interference to be meaningful. If there is a significant change in the phenotype and function of the cells, it is also recognized in the literature. Generally, the interference efficiency can be improved by increasing the transfection efficiency and optimizing the experimental system.

Oligo Synthesis - Beijing Tsingke Biotech Co., Ltd.

What customization options are available for custom RNA oligos?

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The entire process—from synthesis and QC to packaging—is highly customizable. In addition, we offer end-to-end solutions for small nucleic acid screening and delivery, including targeted delivery systems. Our services support high-throughput screening and provide comprehensive analyses such as mRNA/protein silencing efficiency, stability, off-target effects, immunogenicity, and cytotoxicity. The results are reliable and highly reproducible, helping you identify candidate molecules with superior silencing efficiency.

What types of chemical modifications can be incorporated into synthetic RNA oligonucleotides?

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Why is a positive control important in RNA interference experiments?

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What are the maximum absorption and emission wavelengths of commonly used RNA fluorescent dyes?

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Precautions for using siRNA

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How should siRNA be dissolved and stored?

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