Category Archives: Methods

Growing E. coli for the plasmid isolation

  • Amplification of plasmid is desirable for many applications including gene cloning, DNA sequencing, transfection, and probe preparation. Fastest and routinely used method to amplify plasmid is to introduce plasmid in an appropriate strain of E. coli e.g. DH5α (the process is called transformation), grow them to a suitable culture volume, and finally, extract plasmid from them (the process called plasmid isolation).
  • Alternatively, E. coli DH5α harboring a plasmid can also be revived from the stored stocks e.g., glycerol stock or stab culture, if available. Cells obtained from stored stock can be either streaked or plated on an antibiotic containing solid LB-agar plate.
  • Plasmid copy number and culture volume are the two most important parameter which predicts the quantity of the plasmid extracted at the end of the isolation process. Comparatively, large culture volume is required for low copy number plasmid.
  • Plasmid copy number can be increased by chloramphenicol treatment. Several rich growth media can also be used to grow bacteria. These media support high cell density due to nutrient enrichment.
  • Depending on the initial culture volume, plasmid isolation methods are called miniprep (1-5 ml culture volume), midiprep (25-50 ml culture volume), and maxiprep (100-500 ml culture volume).
  • Generally miniprep yields sufficient amount of plasmid for applications like the screening of clones for the presence of insert, DNA sequencing etc. Other applications, like probe preparations, plasmid distribution, transfection, etc., can require a large quantity of plasmid (midiprep or maxiprep).
  • For miniprep, a single colony from the LB-agar plate is inoculated into a antibiotic-containing liquid medium. Culture is grown at 37°C in a shaker incubator overnight (12- 16 h). Grown culture corresponds to late log phase/early stationary phase of bacterial growth and is characterized by low content of RNA. Incubating culture for a long time can cause the death of bacteria, which can result in low yield of plasmid. Sometimes, a well-grown colony from the LB-agar plate can directly be utilized for plasmid miniprep.
  • For a large amount of culture which is required for midiprep and maxiprep, initially, a starter culture is prepared by inoculating a small amount of culture medium (2 – 10 ml) with a single colony. When the culture reaches mid- to late-exponential growth phase (takes 8 – 12 h), culture is diluted in a ratio of 1:100 to 1:1000 to prepare large culture volume for midiprep and maxiprep.
  • All plasmid vectors carry at least one antibiotic resistance gene, which enables bacteria to survive and grow in presence of a respective antibiotic. Antibiotic functions as a selective marker which allows growth of only plasmid containing E. coli cells. In absence of antibiotic, bacteria will lose the plasmid, which will result in low or no yield.

Related Notes:

Protocol – Growing liquid culture of E. coli for plasmid miniprep

Ribonuclease A (RNase A) from bovine pancreas

Overview

  • Ribonuclease A (RNase A) belongs to an endoribonuclease class of ribonucleases. In contrast to exoribonucleases which cleave/degrade RNA in 3’-5’ direction, endoribonucleases degrade RNA endoribonucleolytically in 5’-3’ direction.
  • RNase A is a digestive enzyme which is secreted by the pancreas to digest RNA. It is abundantly present in the pancreas, therefore, the pancreas is a valuable source for RNase A.
  • Mature bovine pancreatic RNase A only has 124 amino acids with molecular weight 13.7 kDa. It lacks tryptophan amino acid.
  • In contrast to others known members of endoribonuclease, RNase A is not a glycoprotein.
  • RNase A is active under a wide range of reaction conditions (temperature range 15 – 70 °C; pH range 6–10). The optimal temperature for its activity is 60 °C and optimal pH is 7.6.
  • RNase A is quite stable to both heat and detergents.
  • It cleaves both single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids at a low salt concentration (0 to 100 mM NaCl). However, it specifically cleaves single-stranded RNA at higher salt concentration (0.3M NaCl or higher)

Cryopreservation of cell culture

  • Cell culture is not static. Cells in culture acquire changes which can be either genetically programmed (e.g., senescence in primary culture) or due to accumulation of genetic abnormalities (mutations, gain or loss of whole chromosomes or part of chromosomes). In addition to this, changes in gene expression pattern and epigenetic modifications due to several reasons including fluctuations in culture condition, contamination, mishandling and stressful condition to culture, can also lead to permanent changes in cell behavior (e.g., stem cell culture can differentiate, or lose its ability to differentiate). Therefore, we need a method to preserve cell culture which stop or slow down these processes.
  • Cryopreservation is an efficient way to preserve cells at ultra-low temperature (below -135°C) which stop all physiological processes and biological aging. It is a routinely used technique in all cell culture laboratories.
  • During preservation at ultra-low temperature, cells die due to many reason including lysis due to ice crystal formation, pH change, dehydration, and alterations in the concentration of electrolytes. Four distinct phases of cell preservation and revival process can cause to damage to cells…………
    • when temperature reduced to above freezing point (hypothermia)
    • when temperature reduced to below freezing point
    • during frozen state
    • during revival
  • Cryopreservation methods ensure that cells are alive at ultra-low temperature and maintain their features when revived after long term frozen state.
  • Most cryopreservation methods rely on
    • cryoprotectants
    • slow cooling
    • rapid revival
  • To cryopreserve cells, cells are suspended in freezing medium, followed by slow cooling and subsequently storage in liquid nitrogen.
  • Freezing medium is nothing but growth medium supplemented with cryoprotectant. Serum containing growth medium contains high amount of serum (upto 90%).
  • Cryoprotectants, the most important component of freezing medium, function by preventing the formation of ice crystals, thus protect cells from lysis.
  • Polyalcohols (e.g., glycerol, ethylene glycol, 2,3 butanediol) and DMSO can be used as cryoprotectants, often a concentration varies from 5 – 20%. Most cryoprotectants have ability to penetrate the cell membrane and function by replacing part of the water in the cell.
  • DMSO is most frequently used cryoprotectant. However, some cells lines are sensitive to DMSO. In such situation, glycerol can be  used. Glycerol is less toxic than DMSO, however, osmotic problem associated with glycerol at the time of thawing restrict its uses.
  • High concentration of serum can also be added in freezing medium. High serum concentration correlate with better survival upon thawing.
  • Serum-free chemically defined freezing medium are also available which are prepared by adding cryoprotectant to serum-free chemically defined medium growth.
  • Serum-containing freezing mediums are used for cell lines growing in serum-supplemented growth medium whereas serum-free freezing medium is used for those cell lines which are maintained in serum-free chemically defined medium.

Cell culture

  • Cells are the basic structural and functional unit of life. Cells from all organisms including multicellular organisms can perform all physiological functions independently. Therefore, cells can be maintained under artificial environment if the right condition is provided.
  • Cell culture can be defined as a process of maintaining cells under the artificially controlled environment in a culture dish, outside their natural environment. This definition can be applied to any organism including prokaryotes, as well as unicellular and multicellular eukaryotes. However, in practice, the term cell culture is used for cells from multicellular organisms, specially multicellular animals. Specific terminology, like bacterial culture (maintaining bacteria in the controlled laboratory environment), yeast culture (maintaining yeast in the controlled laboratory environment), plant culture are used frequently to denote other types of culture.
  • Cells in culture behave as an independent unit like unicellular organisms and perform all necessary functions including cell division and metabolism in the culture dish.
  • Classically, the term ‘Tissue culture’ was used to grow plant and animal explant in a controlled artificial environment in the laboratory. The term ‘Animal tissue culture’ refers to cell culture derived from multicellular animals whereas ‘Plant tissue culture’ refers to the culture of plant cells/tissues.
  • Culture condition must maintain cell’s characteristics as it possesses in its natural environment. Practically, it is very difficult, in part due to limited knowledge of physiological requirement of specific cell type. However, many different cell types have been maintained in culture and are in use in both basic and applied research. One such example is a successful maintenance of stem cell (embryonic and adult stem cells) in culture.
  • Cell culture can be classified based on cell’s characteristics including growth mode, lifespan, morphology and cell types.
  • Based on growth mode of cells, culture can be broadly classified into two types – suspension culture and adherent culture. Semi-adherent culture, which contains loosely adherent cells to the dish surface, also exist.
  • Based on cell morphology in the culture dish, cell culture can be broadly classified into three types – Fibroblast-like, Epithelial-like, and Lymphoid-like.
  • Cell culture is not static. Cells in culture acquire changes which can be genetically programmed (e.g., senescence in primary culture) or due to the accumulation of genetic abnormalities (mutations, gain or loss of whole chromosomes or part of chromosomes). Furthermore, in response to fluctuations in culture condition, cells in culture can show altered behavior due to changes in gene expression pattern which sometimes lead to permanent changes in cell behavior (e.g., stem cells can differentiate).
  • Cell culture technology has found wide application both in basic research and applied research including industry (pharmaceuticals, medical sciences, cancer research, diagnostics, drug and product development, manufacturing of biological compounds, etc.)

Protocol – Cryopreservation of cell culture growing as monolayer in serum-containing medium

Overview

  • Cryopreservation is an efficient way of preserving cell culture. Preserved cells can be revived whenever needed.
  • Cryopreservation not only stop the biological time and aging but also protect cell culture from accidental loss due to mishandling and contamination.
  • Cryopreservation involves freezing of cell suspension at ultra low temperature below -135°C.
  • Cell suspension is prepared in specialized medium, the freezing medium, which contains DMSO and high concentration of serum.
  • DMSO, a cryoprotectant, protects cells from cold shock by preventing ice crystal formation. High serum content also protects cells from death during cryopreservation and revival.
  • Cell suspension is subjected to slow cooling overnight at -80°C before storing them in liquid nitrogen. Slow cooling reduces the probability of of ice crystal formation, thus protects cells from lysis.
  • Serum containing cryopreservative medium is used to preserve cells lines which are maintained in serum containing growth medium.

Requirement

  • Reagents
    • Freezing medium (Composition: 50% FBS + 10% DMSO + 40% growth medium)
    • Complete growth medium (e.g., DMEM supplemented with 10% FBS)
    • PBS without Ca2+/Mg2+
    • Trypsin EDTA (TE)
  • Equipment and disposables
    • Sterile cryogenic vials
    • Sterile conical tubes (15-mL or 50-mL)
    • Controlled rate freezing apparatus
    • Haemocytometer/Trypan Blue
    • -80°C freezer
    • Liquid nitrogen storage container/-150°C freezer
    • Benchtop centrifuge with 45° fixed-angle or swinging-bucket rotor (e.g., Eppendorf™ 5804 Series)
    • Personal protective equipment (sterile gloves, laboratory coat, Full-face protective mask/visor)
    • Laminar flow hood
    • Pipette tips and pipetman
    • Serological pipettes and Pipetboy
    • Inverted phase contrast microscope

Starting materials:

  • Late log-phase monolayer culture (80% confluent), growing in culture dish/flask
Note:
  • Visually inspect culture carefully under an inverted phase contrast microscope and make sure that cells are healthy and do not have any contamination.
  • Properly label Cryogenic vials. You must write cell line name, passage number and date.

Overview of procedure:

Harvested cells from monolayer culture is resuspended in ice-cold freezing medium at the recommended cell density (2 x 106 – 5 x 106). Cell suspension is aliquoted in ice-cold Cryogenic vials (1 ml/vial). All Cryogenic vials are subjected to slow cooling (1 – 3°C/min) overnight in -80°C freezer. Next day, all cryogenic vials are transferred to liquid nitrogen containers or -150°C incubator.

Here we describe a general procedure for cryopreserving adherent cell culture. For specific details, we recommend you to carefully read the manual provided with the cell line.

Protocol

Step 1: Harvest cells from the culture using standard procedure (trypsinization)
  • Discard the culture medium and wash the monolayer with PBS. Add sufficient amount trypsin EDTA solution (1 -2 ml for T25 flask) and incubate at 37°C for 1 – 2 min. Inspect the dish for cell detachment. Incubate at 37°C for some more time if cells are not dislodged.
  • Once the cells are dislodged, tap the flask/dish 2-3 times to make sure all the cells have come out from the dish surface. Add serum containing complete medium (4 ml for T25 flask) and flush the entire surface of dish by pipetting to collect all cells from the dish.
  • Transfer cell suspension to a sterile centrifuge tube. Cells from two or more dishes from the same passage number (subculture) can be combined in one tube.
Note:
  • Serum in the complete growth medium has trypsin inactivating activity.
Tips:
  • At this point, cells can be collected by centrifugation and cell pellet can be resuspended in sufficient amount of complete medium. This step is not necessary. In case of high cell death during trypsinization process, or if the cells are too diluted in suspension, centrifugation and resuspension of cells can be done.
Precautions:
  • Make sure that trypsinization of cells does not cause enormous cell death. A healthy culture is a prerequisite for cryopreservation.
Step 2: Determine viable cell density in cell suspension (optional)
  • Determine viable cell number using trypan blue exclusion assay and hemacytometer.
  • To count viable cell number, mix 20 µl cell suspension to 20 µl trypan blue solution. Place the solution onto haemocytometer chamber.
  • Count live and dead cells. Calculate viable cell count per ml and percentage of dead cells.
Note:
  • You can use other sophisticated methods like Countess® Automated Cell Counter or Moxi Flow Kit which can determine viability cell count quickly.
Tips:
  • If cells are very diluted, collect cells by centrifugation and resuspend cell pellet in appropriate amount of complete medium.
  • In many cases, based on previous experience you can determine how many cryovials can be frozen from a culture dish. One can prepare 2- 4 ml cell suspension in freezing medium from a near confluent T25 flask which can be used to prepare 2 – 4 cryogenic vials (1 ml / vial).
  • Counting is required when cell number is limited and you want to freeze as many cryogenic vials as possible e.g., primary culture.
Precautions:
  • Make sure that cells are properly resuspended before counting viable cells.
Step 3: Resuspend the cells in ice-cold freezing medium at the recommended viable cell density  (2 x 106 – 5 x 106 cells/ml)
  • Harvest cells from cell suspension by centrifugation at 4°C for 5 – 10 min at 250 × g (1000 – 1500 rpm for Eppendorf™ 5804 Series benchtop centrifuge).
  • Carefully aspirate supernatant completely without disturbing the cell pellet.
  • Flick the tube with your finger several times to dislodge the pellet.
  • Add appropriate amount of ice cold freezing medium to obtain right viable cell density (between 2 x 106 – 5 x 106 cells/ml). Resuspend the cells thoroughly with gentle pipetting.
Tips:
  • Cell density in freezing medium can vary with cell lines. In most cases, high cell density is good for cell recovery.
Precautions:
  • Since DMSO can be toxic to cells, it is advisable to use chilled freezing medium. Try your best to maintain the temperature of cell suspension 4°C.
  • Quickly resuspend pellet in freezing medium immediately after aspirating supernatant.
  • Centrifugation speed should be sufficient to get soft pellet. Pellet should not be too tight. Tight pellet will be difficult to resuspend and attempts to resuspend it by vigorous pipetting may cause cell death.
Step 4: Aliquot cell suspension to cryogenic vials
  • Place cryovials on ice.
  • Transfer 1 ml aliquots of cell suspension into cryovials.
  • Tighten caps on vials.
Precautions:
  • While aliquoting, frequently and gently mix the cells to maintain a homogeneous cell suspension.
Step 5: Subjected cryovials to slow cooling (1 – 3°C/min) overnight in -80°C freezer
  • Place all cryogenic vials in controlled rate freezing apparatus (e.g., CoolCell® Cell Freezing Containers from Biocision) and immediately store in -80°C freezer overnight.
Tips
  • The most efficient way to freeze cryogenic vials is to use controlled-rate freezers (e.g., CryoMed™ Controlled-Rate Freezers from ThermoFisher Scientific). However, for most serum grown cell lines, one can use commercially cooling devices e.g., CoolCell® Cell Freezing Containers from Biocision, or  Mr. Frosty™ Freezing Container from ThermoFisher Scientific.
  • Homemade cooling devices – Thermocol box (small size) filled with cotton or tissue paper, can also used in place of commercial cooling devices.
Step 6: Store in liquid nitrogen
  • Transfer all frozen cryogenic vials to a liquid nitrogen container next day. Alternatively, frozen cryogenic vials can also be stored in -150°C freezer.
  • Cryogenic vials can be store in gas phase or liquid phase of liquid nitrogen.
Tips:
  • Revive a vial after 2 weeks to ensure that frozen cells are viable and free of any contamination.
Precautions:
  • Biosafety level 2 cell lines should be stored in the gas phase of liquid nitrogen.
  • You must maintain the proper records of location of frozen cell lines.
  • Wear protective equipments when handling liquid nitrogen. Remember that cryogenic vials may explode if they are stored in liquid phase of liquid nitrogen.

 

Bacterial contamination in cell culture

  • Bacterial contamination is one of the most common cell culture contamination.
  • Poor aseptic culture condition, including handling, incubator and laminar flow hood, or culture media, can be common source of bacterial contamination.
  • Contaminated culture often becomes turbid and the medium turns yellow (phenol red containing medium).
  • Microscopic inspection of such culture is often sufficient to confirm the presence of bacteria. Motile bacteria and bacterial clumps are often observed in contaminated culture, which can easily be distinguished from cell debris. However, low level of contamination may go undetectable in the presence of antibiotics in culture.
  • In such cases, suspected contaminated culture can be grown in absence of antibiotics, which will allow the bacteria to grow, thus contamination can be detected easily.
  • Usually a contaminated culture once confirmed is discarded immediately. In case of precious culture, one can try to rescue the culture by treating the culture in presence of high concentration of antibiotics. Frequently replacing the culture medium containing high concentration bactericidal antibiotics may help to eliminate the bacterial contamination.

Stock solution

Overview :

  • Stock solution can be defined as a highly concentrated solution, mostly prepared as 10X concentrated, which can be diluted to prepare working solution.
  • Stock solution is also used as a component to prepare complex solution containing many ingredients. For example, stock solution of EDTA (0.5 M EDTA solution) can be used to prepare many solutions including TAE, TBE, Tris-EDTA, Trypsin-EDTA etc.

Advantages :

  • Since stock solution is highly concentrated, its storage requires less space.
  • It is also convenient to transport stock solution due to its small volume.
  • The concentration of various component of working solution is more accurate when it is made from stock solution rather than individual component from its original form.
  • Stock solutions which contains any active biological substances (e.g., enzymes, inhibitors, DNA, RNA etc.) are more stable in highly concentrated form.

Disadvantages :

  • Since stock solution is highly concentrated, often to dissolve them requires more time and effort. Sometime, heating of solution is required to dissolve the substance completely.
  • Long term storage of stock solution (especially salt solution, at cold room) may leads to precipitation of solute.

Comparison of TAE and TBE electrophoresis buffer

Properties TBE TAE Remarks
Buffering capacity High Low TAE become exhausted during extended or repeated electrophoresis
Migration of (double stranded) DNA Slow Fast TAE has better conductivity
Resolution High resolution long DNA fragments High resolution for Short DNA fragments TBE supports agarose cross-linking better than TAE
Enzymatic modification of gel purified DNA Poor Good Borate from TBE buffer is an inhibitor of many commonly used enzymes in molecular biology
Recovery of DNA from agarose gel Poor Good Borate from TBE buffer interacts with DNA
Integrity of DNA High Low Borate from TBE buffer inhibits many enzyme activity including DNA modifying enzymes
Cost Costly Cheaper Highly conc. TAE (50X) require less volume to be transported.
Active buffer Tris – Acetate Tris – Borate
Working conc for DNA electrophoresis 1X 0.5X

 

DNA loading dye

  • DNA samples are mixed with DNA loading dye / buffer prior to loading into the wells of agarose gel for electrophoresis.
  • Usually a DNA loading dye contains at least one dye (orange G, bromophenol blue, xylene cyanol FF or bromocresol green) and a high density reagent (glycerol, sucrose or Ficoll 400).
  • EDTA and/or SDS can also added in the loading dye.
  • Ingredients are dissolved in water, but tris buffer can also be used in place of water. Tris containing DNA loading dye is called DNA loading buffer.
  • DNA loading dye/buffer serves the following purposes_ _ _ _
    • It provides high density to DNA sample. Due to high density, DNA sample settled at the bottom of the agarose wells. Nicely settled DNA form sharp and crisp band upon electrophoresis.
    • DNA loading dye/buffer imparts colour to DNA sample which allows easy monitoring of sample loading process. Sample overflow, cross-contamination, or leakage of wells can easily be monitored due to colour which otherwise would be very difficult with colourless sample.
    • Since dye color can be seen by naked eye, electrophoresis progression can be easily monitored by observing the migration of dyes which otherwise would be difficult because DNA migration can not be monitored by naked eye and DNA visualization requires staining and special instrument (ethidium bromide staining and uv torch).
  • There are several dyes (Bromophenol blue, Bromocresol green, Orange G, Xylene cyanol FF) which can be used for the preparation of DNA loading dye/buffer. An ideal dye, used for preparation of DNA loading dye/buffer, should have following characteristics.
    • It should not interact with the DNA or component of electrophoresis buffer and gel.
    • Like DNA, It should have negative charge and move towards the positive pole.
    • It should imparts contrast colour to DNA sample and its migration should easily be monitored visually.
    • It should not interfere with the analysis of DNA.
    • The size of the dye must be within the upper and lower resolution limit of the agarose gel (50 bp to 20 KB)
  • Two dye containing loading buffer (mostly bromophenol blue and xylene cyanol FF) is very common for DNA gel electrophoresis. In such loading buffer, One dye migrates fast and correspond to the migration of DNA fragment 50 – 500 bp, whereas other dye migrate comparatively slow and correspond to the migration of DNA fragment 4000 – 8000 bp.
  • Use of a specific dye mostly depends on the application and the size of the DNA fragment(s) to be analyzed by the gel electrophoresis. DNA band can be masked by co-migrating dye, which can give wrong information about the amount of DNA fragment. Depending on the size of DNA fragment, one should choose the dye.
  • The high density reagent (glycerol, sucrose or Ficoll 400) is added to facilitate the sample to be placed in the well of the gel. Due to high density, DNA sample settled at the bottom of the well. It also helps DNA sample to be confined in the well without diffusing out.
  • Since glycerol interacts with the borate of TBE electrophoresis buffer, glycerol containing loading dye / buffer are not recommended to use with TBE buffer.
  • Sucrose containing loading dye / buffer are prone to develop mold like growth. Therefore, sucrose containing loading dye / buffer can not be kept for long time at 4°C.
  • Ficoll-400 containing loading dyes / buffers can be stored at room temperature and also function as better density agent.
  • EDTA is added to inhibit the action of nucleases or DNA modifying enzymes which require divalent cation for their action.
  • Tris functions as a buffering agent and maintain the pH of the loading dye.
  • SDS reduces the DNA-protein interaction, thus helpful to run DNA samples containing proteins or enzymes (e.g., alkaline phosphatase).
  • DNA loading buffer is generally made in 6X concentration and mostly contains two dyes, bromophenol blue and xylene cyanol. Some DNA loading dyes contain only one dye while others may contain three dyes.

Tracking dyes

  • Tracking dyes are coloured dyes, which are added to the loading buffer/solution.
  • Loading dye/buffer are used to prepare sample for gel electrophoresis.
  • Tracking dyes serve two purposes:
    • They impart colour to the sample, thus visualizing the sample loading process.
    • Since they are visible by naked eye, the progression of gel electrophoresis can easily be monitored.
  • Tracking dyes should have the following properties in order to be used for gel electrophoresis:
    • They should not interact with the components of sample (DNA or protein).
    • They should move from negative to positive electrode (the direction of electrophoresis). Therefore, they should have net negative charge at pH of electrophoresis buffer.
  • Common tracking dyes used for gel electrophoresis: Bromophenol blue, Xylene cyanol FF, Orange G