Because the purity of the primer cannot be 100%, it is possible to select a clone that has been amplified by the impurity primer during the selection of the clone. At this time, please re-select a clone for sequencing, and you will get the correct result. If the sequencing results of 2 ~ 3 clones have not improved, we will re-synthesize the primer for free and deliver it to you as soon as possible. If you file a claim, according to international industry practice, the claim scope is limited to the price of the product.

EB stains DNA by intercalating between the base pairs of a double helix. Synthesized DNA molecules are single-stranded and can only be stained by EB if they form local hairpin loops or partial double-stranded structures by folding back on themselves. Since the sequences of different primers are different, their ability to form double helices is also different. Therefore, the brightness of the EB-stained bands cannot be used to quantify the synthesized DNA.

PCR reactions can fail for a variety of reasons. Here are some of the most common causes:Primer-template annealing: The primers must be complementary to the template sequence in order to anneal properly. If the primers do not anneal, the PCR reaction will not be able to start.Primer structure: The primers should not have any secondary structures that could interfere with annealing.Reagent quality: The PCR reagents must be of high quality in order to function properly.PCR instrument: The PCR instrument must be working properly in order to generate the desired results.PCR reaction conditions: Th…

For primer electrophoresis, it is essential to employ denaturing PAGE electrophoresis. As the primers are single-stranded DNA and prone to forming intricate three-dimensional structures, performing Agarose gel electrophoresis can lead to the appearance of multiple bands, making it impractical for quantitative analysis.

Because nucleic acids exhibit strong absorption near 260nm, and proteins show strong absorption near 280nm, the A260/A280 ratio is commonly used to assess nucleic acid purity when extracted from biological samples, with the ratio typically falling between 1.8 and 2.0. This judgment is based on the assumption that the proportions of A, G, C, and T in the sequence are roughly equal. However, for synthesized DNA/RNA, especially with short sequences (usually between 20 and 30 bases), the proportions of A, G, C, and T can vary significantly. Due to the different molar extinction coef…

No, both the 5' and 3' ends have -OH groups. If a phosphate group is needed, modification synthesis is required to introduce phosphorylation.

A convenient method in the laboratory is using the PAGE (Polyacrylamide Gel Electrophoresis) technique. Electrophoresis is performed on a polyacrylamide gel with 7M urea at a specific concentration. For primers with <12 bases, use a 20% gel; for primers with 12-60 bases, use a 16% gel; for primers with >60 bases, use a 12% gel. Take around 0.2 OD of the primer and dissolve it in urea-saturated solution or add urea powder to the primer solution until saturated. Before loading, heat denature the sample (95°C, 2 mins). The addition of urea serves the purpose of denaturation and increases s…

Typically, when synthesizing long-chain primers, the amount of reagents required is significantly higher than for short-chain primers, especially for primers exceeding 90 base pairs. Due to the increased cost of reagents, this results in an overall higher price for the synthesis of long-chain primers.

Sequences up to 150 bases in length can be synthesized.

The rough calculation for the nmol of synthesized oligo is done using the formula:\text{nmol} = \frac{\text{OD value} \times 33 \mu g}{\text{Number of bases} \times 330} \times 1000 = \frac{\text{OD value}}{\text{Number of bases}} \times 100nmol=Number of bases×330OD value×33μg×1000=Number of basesOD value×100For an accurate concentration of synthesized DNA, you can use the formula:\text{nmol} = \frac{\text{OD value} \times 1000}{(15.3 \times A) + (7.4 \times C) + (11.8 \times G) + (9.3 \times T)}nmol=(15.3×A)+(7.4×C)+(11.8×G)+(9.3×T)OD value×1000Where A, C, G, and T represent the re…

Oligos can be quantified using a UV spectrophotometer by measuring the absorbance of the solution at a wavelength of 260nm. It is recommended to dilute the solution to an absorbance range of 0.2-0.8 during measurement (extremely high or low absorbance can lead to significant errors). For DNA powder, it is dissolved in water with thorough shaking, and a portion of the solution is diluted to 1ml for measuring absorbance in a 1ml standard cuvette. The measured OD value for the given volume can then be used to calculate the OD value of the original solution.

Unresolved primers are highly stable and can be stored for at least one year at -20°C. For dissolved primers, it is recommended to pre-dilute them to a storage solution of 100 μM, aliquot into tubes, and store at -20°C. This can preserve them for at least six months or more (repeated freeze-thaw cycles can reduce their shelf life). Before use, dilute the concentrated solution to the working concentration for experiments.

Our synthesis report provides the amount of water to add for diluting each OD of the primer to a concentration of 100 μM (or 100 pmol/μl). For dissolution, you can add an appropriate volume of RNase-free double-distilled water (pH > 6.0) or TE buffer (pH 7.5-8.0) based on your experimental needs. Before opening the vial, it is advisable to centrifuge it at a speed of 3000-4000 rpm for 1 minute to prevent primer loss when the cap is opened.

Purification methods for Aegi primers:1. **C18 Column Desalting:** Also known as a simple reverse-phase column, it has specific adsorption for DNA, which can be eluted with an organic solvent but not with water. This effectively removes salt.2. **R-PAGE (Reverse-Phase PAGE):** Based on specific or reverse chromatography, it removes failed sequences from the synthesized product, suitable for primers below 35 bases.3. **PAGE Purification:** Using denaturing polyacrylamide gel electrophoresis, it separates primer DNA and then recovers the target DNA from the gel.4. **HPLC Purification:** Using th…

Primers are synthesized using the solid-phase phosphoramidite method. Various DNA synthesizers exist, but regardless of the machine used, the synthesis principles remain the same. The main differences lie in the synthesis yield, reagent consumption, and the time taken for a single cycle.1. **Deprotection:** Addition of deblocking reagent to remove the protecting group (DMT) from the 5'-OH of the base, obtaining a free 5'-OH.2. **Coupling:** Simultaneous addition of an activator and a new nucleotide. The 5'-OH of the new base remains protected by DMT, and a coupling reaction occurs …

Some cells are exceptionally large or small, especially certain primary cells, and some cells aggregate, making counting difficult. The approach for these cells may vary from the preliminary experiments. During the pilot experiment, maintain a cell confluence of 30-40% at the time of infection. Even if the cells do not reach the predefined count of 10,000 cells/well, try to control the cell confluence around this quantity for the main experiment. For cells that form clusters, such as pancreatic islet cells or embryonic stem cells, pay attention to the number and size of cell clusters for futur…

Lentivirus may exhibit some toxicity towards your target cells. Adjust and reduce the MOI (Multiplicity of Infection) during infection. Additionally, consider changing the medium at 4 hours, 8 hours, and 12 hours post-infection with fresh complete culture medium for continued observation and cultivation.

The GFP fluorescence intensity in target cells depends on several factors, including the number of virus particles infecting the cells, the proliferation status of the cells, cell type, and the observation time. Generally, the more virus particles infect the target cells, and the faster the cells proliferate, the stronger the GFP fluorescence will be. Lentivirus is a slow virus, and typically, the peak expression of the GFP gene is reached 48-72 hours after virus infection in rapidly proliferating cells. For cells with slower proliferation rates, the expression time of the GFP gene may be exte…

Typically, we increase the MOI (Multiplicity of Infection) to enhance viral transduction efficiency. When necessary, Polybrene (4-10 µg/mL) can be added to the culture medium to increase the infection rate of the virus. Additionally, ensuring optimal growth conditions for the target cells is crucial for achieving a normal infection efficiency.

The recommended MOI (Multiplicity of Infection) for common cell types can vary and is influenced by factors such as cell growth status, the type of infecting virus, and experimental conditions. Generally, the recommended MOI range is between 0.5 to 5. However, the optimal MOI may need to be optimized based on specific experimental conditions.Here are some recommended MOI ranges for common cell types, but please note that these are only references, and it is advisable to adjust based on specific experimental conditions:1. **HEK293 cells:** 1-52. **HeLa cells:** 1-53. **THP-1 cells:** 5-104. **H…

When the MOI value required for cell infection reaches 80%, it is defined as the optimal MOI value for these cells.Polybrene: Commonly used infection additive at a concentration of 5~10 µg/mL. Polybrene is a positively charged small molecule that binds to the anions on the cell surface, significantly enhancing virus-cell contact and increasing virus infection efficiency. It can improve infection efficiency by 3~4 times for general cells and up to 10~20 times for certain cells.1.1 Experiment12-well plate, pipette gun, tips, EP tubes, Polybrene, HBSS, waste liquid1.2 Experiment1.2.1 Adherent Ce…

The lentiviral vector provided by Aigi belongs to the "second generation" lentiviral vector. The enhancer function of the 3' LTR in its genome is deleted, forming the so-called "self-inactivation" (SIN). After the viral genome integrates into the cell genome, it does not produce new progeny viruses, reducing the risk of unintended activation of surrounding genes, thus enhancing safety. However, the virus still carries potential biological risks, so it is recommended not to use pseudotyped viruses encoding known or potentially carcinogenic genes. Unless a gene is fully r…

1. Upon receipt, store the virus solution at 4°C (please use within one week). For long-term storage, keep it at -80°C. Avoid repeated freeze-thaw cycles as it may reduce virus titers; generally, viruses can be stored at -80°C for approximately six months. If stored for more than six months, reevaluate the virus titer before use.2. Lentivirus provided by Aigi is dissolved in HBSS (pH 7.4). When using the virus, thaw it in an ice bath after retrieving it from the -80°C freezer and store it at 4°C. If dilution is required, add an appropriate amount of HBSS for uniform mixing, or dilute it w…

How to Ensure the Reliability of Assembly Results?To ensure the reliability of assembly results, in addition to maintaining Contig N50 and Scaffold N50, it's crucial to assess assembly quality. This can be achieved through various methods:1. **EST and RNA Data:** - Evaluate the integrity of assembled genes using EST (Expressed Sequence Tag) and RNA data.2. **BAC Data:** - Check for misassemblies or misjoins using BAC (Bacterial Artificial Chromosome) data.3. **Conserved Gene Evaluation:** - Assess genome assembly completeness through methods like CEGAM or BUSCO, which evaluate the pr…

Translation:**Which species can be studied using WGBS?**To conduct whole-genome methylation analysis, the species should meet the following criteria:a. The species must be eukaryotic.b. The species should have a reference genome, assembled at least to the scaffold level.c. It should have relatively comprehensive annotations.**What are the advantages of Whole Genome Bisulfite Sequencing (WGBS)?**At the whole-genome level, WGBS detects methylation sites at single-base resolution. It not only accurately identifies changes in methylation levels in common regions such as CpG islands but also analyz…

Translation:**Can Small RNA Sequencing be conducted for Species without a Reference Genome?**Small RNA sequencing studies can be conducted for species without a reference genome. However, in such cases, it is necessary to perform de novo transcriptome assembly to generate a transcriptome, which can then serve as a reference sequence for small RNA sequencing analysis.**What does the Preference Analysis Graph of the First and Each Base in Sample miRNA Analysis Indicate?**In miRNA precursor development into mature forms, the process is orchestrated by Dicer enzyme cleavage. The specificity of cle…

**Differences between Chip Technology and Second-Generation Sequencing Technology in Analyzing circRNA:**Chip technology can only detect known circRNAs, and the noise signal is relatively large. Currently, research on circRNAs is relatively limited, and circRNA annotation is not comprehensive. Additionally, circRNA expression has strong temporal and spatial specificity, making it challenging to ensure that the data obtained under experimental conditions match the data in the database. This may result in the loss of a significant number of specifically expressed circRNAs. Second-generation sequ…

**Which Species Can be Studied for lncRNA Research?**To conduct lncRNA analysis, the species must meet the following criteria:a. The species must be eukaryotic.b. The species must have a reference genome, at least assembled to the scaffold level.c. The species should have relatively comprehensive annotations.**Why Remove rRNA During lncRNA Sequencing Library Construction?**In lncRNA, only the 3' end of lincRNA carries a polyA structure, while other lncRNAs lack a polyA structure. Therefore, to obtain a more comprehensive understanding of lncRNA information, it is not recommended to use oli…

**Ion S5 XL Platform for Amplicon Sequencing at Novogene: Advantages and Recommendations**The Ion S5 XL platform utilized by Novogene employs semiconductor sequencing technology, converting chemical signals directly into digital signals on a semiconductor chip. This system operates without laser light sources, optical systems, or cameras. Using unlabeled nucleotides and enzymes for sequencing, it significantly enhances the accuracy of base calling by detecting H+ ions. Compared to the Illumina HiSeq PE250, this platform offers longer read lengths without the need for assembly and shorter seque…

**Differences Between Resequencing and Genome-Based Variation Detection:**In genome-based studies, more variation information can be obtained compared to resequencing, especially in highly variable regions of the genome and for discovering new gene information. Additionally, for strains with distant relationships, the genome-based approach is more ideal due to the presence of numerous highly variable regions. Resequencing, on the other hand, has its main advantage in terms of cost. For evolutionary studies involving a large number of closely related strains, using resequencing results as a fou…