Immunohistochemistry Collagen Type VII Rabbit

Get tips on using TRIzol™ Max™ Bacterial RNA Isolation Kit to perform RNA isolation / purification Bacteria - Gram negative Vibro parahaemolyticus

Get tips on using NA-Star™ Influenza Neuraminidase Inhibitor Resistance Detection Reagent Set to perform Cell Culture Contamination Detection Kit Virus

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - U937 MK2 (MAPK Kinase 2) Viral vectors

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - Jurkat MK2 (MAPK Kinase 2) Viral vectors

Bacterial culture is a process of letting bacteria multiply in a controlled fashion (temperature, humidity, oxygen content or shaking), in a predetermined culture medium (antibiotic resistance to obtain homogenous clones). It is an important step, especially during cloning, as a single cell can be grown homogeneously (on semi-solid or in liquid conditions) to obtain colonies. As mentioned, bacteria can be cultured in broth cultures (Luria broth or LB) or Petri dishes (Agar plates). A specific antibiotic can be added to the broth or agar plates in order to grow bacteria which have the gene insert conferring its resistance to that antibiotic. Following points are necessary to consider for optimal growth conditions: 1. In general, most bacteria grow well at 37C, but there are some strains which require growth temperatures between 25-30C. 2. It is ideal in broth cultures to fill the flask to ⅓ or less of the total flask volume for optimal aerobic growth. 3. Shaking speeds between 140-180 rpm are appropriate to ensure aeration and that the cells are surrounded by fresh media, and do not settle.

Bacterial culture is a process of letting bacteria multiply in a controlled fashion (temperature, humidity, oxygen content or shaking), in a predetermined culture medium (antibiotic resistance to obtain homogenous clones). It is an important step, especially during cloning, as a single cell can be grown homogeneously (on semi-solid or in liquid conditions) to obtain colonies. As mentioned, bacteria can be cultured in broth cultures (Luria broth or LB) or Petri dishes (Agar plates). A specific antibiotic can be added to the broth or agar plates in order to grow bacteria which have the gene insert conferring its resistance to that antibiotic. Following points are necessary to consider for optimal growth conditions: 1. In general, most bacteria grow well at 37C, but there are some strains which require growth temperatures between 25-30C. 2. It is ideal in broth cultures to fill the flask to ⅓ or less of the total flask volume for optimal aerobic growth. 3. Shaking speeds between 140-180 rpm are appropriate to ensure aeration and that the cells are surrounded by fresh media, and do not settle.

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

Get tips on using 300 prep FavorPrep™ Plasmid DNA Extraction Mini Kit (sample size: 1~ 5 ml culture cells) to perform Plasmid Isolation Vibrio parahaemolyticus

Plasmid isolation is an important technique in molecular biology or any kind of genetic editing. It involves amplifying plasmids overnight by transforming them into competent bacterial cells. The desired colonies of these bacteria can then be grown in shaker cultures, at appropriate shaking speed, oxygen availability and temperature. These liquid cultures can then be ultracentrifuged to pellet the bacteria, which are then used for plasmid isolation. The bacteria are first resuspended in a buffer, then lysed, neutralized, purified in a column, eluted, precipitated with ethanol and then resuspended. During plasmid isolation, it is important to lyse cells quickly because lysing bacteria for too long may lead to irreversible denaturing of the plasmid. Usually, alkaline lysis is used for isolation because it is a mild treatment. It isolates plasmid DNA and other cell components such as proteins by breaking cells apart with an alkaline solution. Precipitation removes the proteins, and the plasmid DNA recovers with alcohol precipitation. Resuspension and lysis buffers should be mixed thoroughly in order to prevent the DNA from breaking into smaller fragments. This is because broken gDNA can reanneal and remain in the solution, without binding to the column.

Plasmid isolation is an important technique in molecular biology or any kind of genetic editing. It involves amplifying plasmids overnight by transforming them into competent bacterial cells. The desired colonies of these bacteria can then be grown in shaker cultures, at appropriate shaking speed, oxygen availability and temperature. These liquid cultures can then be ultracentrifuged to pellet the bacteria, which are then used for plasmid isolation. The bacteria are first resuspended in a buffer, then lysed, neutralized, purified in a column, eluted, precipitated with ethanol and then resuspended. During plasmid isolation, it is important to lyse cells quickly because lysing bacteria for too long may lead to irreversible denaturing of the plasmid. Usually, alkaline lysis is used for isolation because it is a mild treatment. It isolates plasmid DNA and other cell components such as proteins by breaking cells apart with an alkaline solution. Precipitation removes the proteins, and the plasmid DNA recovers with alcohol precipitation. Resuspension and lysis buffers should be mixed thoroughly in order to prevent the DNA from breaking into smaller fragments. This is because broken gDNA can reanneal and remain in the solution, without binding to the column.