Make the life of hard-working bench scientists easier and allow them to dedicate more time to their subjects of interest.

Description of  Biocal

Experimental research in molecular biology requires fundamental techniques such as restriction enzyme digestion and DNA ligation, which are essential for recombinant DNA technology and widely practiced in laboratories worldwide. Mastering these techniques is crucial for progress in biological research.

Our app simplifies these processes by providing precise volumes for each component required in restriction digestion and ligation reactions. Users no longer need to calculate DNA volumes manually; instead, they input the desired DNA concentration, and the app calculates the necessary volumes for your reactions. The app then conveniently provides these volumes, so you can quickly set up your reactions ready for incubation at the appropriate temperatures in Eppendorf tubes.

Additionally, our app offers the necessary DNA volumes for DNA assembly reactions, accommodating up to six different DNA fragment assemblies when constructing and using two or more DNA molecules.

This user-friendly app ensures seamless operation, allowing instant use without the need for detailed instructions. It also guides users in sampling volumes to normalize mass and molar concentration across tubes with containing different DNA concentrations and sizes. Furthermore, it provides options for calculating various physical quantities of DNA and RNA of interest.

Please note that all calculations involving DNA experiments are performed on double-stranded DNA molecules in BioCaL.

The app also includes features for normalizing cell optical density to adjust the number of cells taken from different samples, as well as evaluating plasmid DNA copy number per cell, facilitating thorough analysis and efficient experimentation.

 

BioCaL tasks

DNA Restriction Enzyme Digestion:

DNA restriction digestion, or simply restriction digestion, is a technique used to cleave double-stranded DNA molecules at specific sites using enzymes known as restriction endonucleases or restriction enzymes. These enzymes recognize specific DNA sequences (restriction sites) and cleave the phosphodiester bonds within those sequences. The result is the generation of fragments with defined ends determined by the restriction site. The cut sites are often palindromic sequences, meaning they read the same backward as forward. Restriction digestion is a crucial step in many molecular biology applications, such as cloning, DNA mapping, and analysis. It allows for the manipulation of DNA fragments by creating compatible ends for subsequent steps.

     BioCaL provides the volumes of your substrate DNA according to the reaction volume, the concentration of your DNA, your desired mass of DNA, and the enzyme volume. It summarizes the reaction table, ready for pipetting to mix your reaction elements in the Eppendorf tube.

 

DNA Ligation:

DNA ligation is the process of joining two DNA fragments together covalently using the enzyme called DNA ligase. After DNA restriction digestion, the desired DNA fragments are ligated together to create a new DNA molecule. DNA ligase catalyses the formation of phosphodiester bonds between the adjacent nucleotides in the DNA fragments, resulting in a continuous DNA strand, namely without any nicks. DNA ligation is a fundamental step in various molecular biology techniques, including cloning, plasmid construction, and the creation of recombinant DNA molecules. It is crucial for assembling different DNA fragments into larger, functional units.

     BioCaL provides the volumes of your vector DNA (DNA1) and insert DNA of your interest (DNA2) according to the reaction volume, concentration, and length of both DNAs, the mass of your vector DNA you want to use in the ligation reaction, molar ratio of DNA2/DNA1, and ligase volume. It summarizes the reaction table, ready for pipetting to mix your reaction elements in the Eppendorf tube.

DNA Ligation-1: The user can freely choose the vector DNA amount and the insert/vector ratio.

DNA Ligation-2: The user just provides the vector amount, then the insert/vector ratio is determined by the insert and vector size and BioCaL provides the appropriate ligation condition table.

DNA Ligation-3: The user provides the vector amount then BioCaL presents the five ligation conditions, namely insert/vector ratios of 1:1, 2:1, 3:1, 4:1, and 5:1, respectively.

*Please bear in mind these are general ligation conditions: the insert size range is more than 10% and less than 100 % of the vector size. When you ligate double stranded oligos, please refer to your specific protocol.

 

DNA assembly reaction:

DNA assembly reactions are laboratory techniques used to construct DNA molecules from smaller DNA fragments. These reactions are essential in molecular biology and genetic engineering for creating custom DNA sequences, such as genes, plasmids, or entire genomes. There are several methods for DNA assembly, but they generally involve joining DNA fragments together through complementary base pairing. The DNA fragments are mixed together in a reaction tube along with specific enzymes and buffer solutions (assembly buffer) optimized for DNA assembly. These enzymes may include DNA ligase, which catalyses the formation of phosphodiester bonds between adjacent DNA fragments, or DNA polymerase, which can fill in gaps and extend the DNA strands. 

     This app provides the accurate volume of each fragment for the assembly reaction to maintain the vector-to-insert ratio at an optimized level. Specifically, when two to three molecules assemble, the vector-to-insert ratio is 1:2 (molar ratio), and when three to six, the vector-to-insert fragments ratio is 1:1 (molar ratio). The user simply specifies the desired amount of vector in picomoles (pmol) and the fragment length [bp] along with its concentration in [ng/µL] in the assembly reaction. The app calculates the required volume of each DNA fragment with the optimized mole fragment ratio for the assembly reaction.

     In the optimized fragment ratio within the reaction mixture, the overlapping regions of the DNA fragments efficiently anneal together through complementary base pairing. Once the fragments are aligned, DNA ligase catalyses the formation of covalent bonds between the phosphate backbones of adjacent fragments, creating a continuous DNA molecule.

     DNA assembly reactions are highly versatile and can be used for various applications, including gene cloning, genetic circuit construction, and synthetic biology projects. Different DNA assembly methods offer advantages in terms of efficiency, accuracy, and scalability, allowing researchers to choose the most suitable technique for their specific needs.

 

Normalisation by mass concentration [ng/µL]:

When you wish to sample the same DNA mass from multiple tubes containing double-stranded DNA with varying amounts, BioCaL provides the sampling volume for each DNA and water to prepare your desired sampling mass concentration of each DNA. By using BioCaL, you can easily prepare the same mass concentration for all of your samples. You might want to use this function to normalize the DNA mass when transforming DNA into the cell.

 

Normalisation by molecular concentration [fmol/µL]:

When you wish to sample the same DNA molecular number from multiple tubes containing double-stranded DNA with varying amounts, BioCaL provides the sampling volume for each DNA and water to prepare your desired sampling mole concentration of each DNA. By using BioCaL, you can easily prepare the same molecular number concentration for all of your samples. You might want to use this function to normalize the DNA molecular number when transforming DNA into the cell.

 

Mass to moles or moles to mass conversion:

Feed your DNA mass [ng] and length [bp] to BioCaL to convert to moles [pmol] or moles [pmol] to mass [ng].

 

OD260 to mass concentration conversion:

You can simultaneously determine ssRNA, ssDNA, and dsDNA concentrations in [ng/µL] by inputting your OD260 reading into the app.

 

DNA physical quantities:

Feed your DNA mass  [ng] and length [bp] into BioCaL; it converts the mass [ng] to moles [fmol] and provides related information such as molar mass [g/mol], DNA copy number in the mass, unit DNA mass [ng], volume [nm^3], and length [mm].

 

RNA physical quantities:

Feed your RNA mass  [ng] and length [bp] into BioCaL; it converts the mass [ng] to moles [fmol] and provides related information such as molar mass [g/mol], RNA copy number in the mass, unit RNA mass [ng] and unit charge in atto-coulomb [aC] .

 

Plasmid copy number evaluation:

A plasmid is a small, circular DNA molecule found in bacteria that can replicate independently from the bacterial chromosome. Plasmids can exist in two main types: stringent and relaxed. Stringent plasmids typically have a low copy number, ranging from 1 to 5 copies per bacterial cell, while relaxed plasmids have a higher copy number, often ranging from 10 to 100 copies per cell. Plasmid isolation is routine work in the wet lab. This option evaluates the copy number of your plasmid in the bacterial cell by providing the initial culture volume, its optical density, the OD600 to CFU/mL conversion factor, and the total mass of your isolated plasmid and its length, namely the ratio between the total copy number of your isolated plasmids and the total number of cells utilised for plasmid isolation. Understanding the copy number of your plasmid is crucial for various genetic engineering applications and experimental designs.

 

Cosmid packaging efficiency:

A cosmid is a type of cloning vector used in molecular biology for the cloning and manipulation of large DNA fragments. It is essentially a plasmid vector that contains the cos site from lambda phage DNA, which allows it to be packaged into lambda phage particles. Cosmids are particularly useful for cloning large DNA fragments because they can carry inserts much larger than those carried by conventional plasmids.

In vivo packaging in a helper strain refers to the process of packaging the cosmid DNA into lambda phage particles inside a bacterial host cell that provides the necessary machinery for packaging. The helper strain typically lacks its own lambda phage genome but carries genes required for phage DNA packaging, such as genes encoding phage structure proteins, head and tail proteins. This enables the cosmid DNA to be packaged into lambda phage particles within the bacterial host cell.

     The efficiency of cosmid packaging can be evaluated by various methods, such as  blue/white screening, rescue assay and fluorescence-based assays etc. In BioCaL, using the total cosmid transduction unit obtained from those assays to evaluate the packaging efficiency.  For example in the rescue assay approach, the packaged cosmid DNA is isolated from the lysed bacterial cells and then transfected into a recipient E. coli strain lacking the cosmid. Successful rescue of the cosmid allows the recipient cells to grow on selective media, enabling the quantification of the rescued cosmid and thus the assessment of packaging efficiency, namely the ratio between the cosmid transduction cells and the total helper cells used in vivo packaging.

 

Restriction enzyme finder:

The newly added restriction enzyme finder offers users a quick and convenient way to retrieve information about specific restriction enzymes. Users can enter either the enzyme name to access its recognition sequence or input the recognition sequence to identify matching enzyme(s). Enzymes are colour-coded based on their cutting end structure: blue for 5'-protruding, green for blunt, and red for 3'-protruding ends. The app also provides Type II subtypes, A(symmetric), P(alindromic) and S(hifter) based on their recognition sequence and cleavage properties. Type IIA enzymes typically recognize asymmetric sequences and cleave at or near their recognition sequence, producing defined DNA fragments. Type IIP enzymes recognize palindromic sequences and cleave at or near their recognition sequence. Type IIS enzymes typically recognize non-palindromic sequences and cleave at a defined distance shifted away from the recognition site. Each enzyme entry includes a link to REBASE for more detailed information on the selected enzyme. This feature simplifies the process of selecting suitable enzymes for molecular biology experiments, thereby accelerating research and experimentation.

Note: When entering enzyme names, such as "III" in "HindIII," remember that "III" uses Roman numeral characters ("I" pronounced as "ai"), not the lowercase letter "l" ("el") or the numeral "1" ("one").