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Sunday, March 31, 2013

List of Biotech companies in Hyderabad

  • Albany Molecular Research Centre Pvt Ltd. Hyderabad
  • Anshul Biotechnologies Aurobindo Pharma Ltd
  • Biomax Life Sciences BioGenex
  • EPR Pharmaceuticals Ltd EPR
  • GVK Biosciences Pvt Ltd
  • Helvetica Industries Pvt. Ltd
  • Indigene Pharmaceuticals Ltd
  • Laurus Laboratories Ltd
  • LAXAI AVANTI Life Sciences
  • Mithros Chemicals
  • Nektar Therapeutics India
  • Pioneer Overseas Corporation
  • Regain Biotechnology
  • Sai Life Sciences
  • United States Pharmacopeia (USP)
  • Indian Institute of Chemical Technology
  • Centre for Cellular and Molecular Biology
  • Vivimed Labs Ltd

Sunday, March 24, 2013

Real-time PCR inhibition and poor reaction efficiency

Real time PCR efficiency is one of the critical factor in real time PCR assay development and optimization. A 100% efficient PCR result indicates a well optimized assay development. Below which indicates a need to further optimize the reaction parameters, which includes, to mention few, primer probe concentration, buffer optimization, etc. An accepted PCR assay efficiency ranges from 90% - 110%. A standard curve can be generated andefficiency can be calculated.


Real - time PCR: Causes of high or low efficiency



As mentioned earlier an accepted PCR assay efficiency range for real time PCR assay is 90% - 110%. An assay efficiency above 110% indicates a possible inhibition in the real time PCR reaction. The main reasons for inhibition in the reaction is the poor quality of DNA or RNA used as template, use of high template concentration. Sub-optimized extraction procedures to get the DNA or RNA purified, presence of high amount of chaotropic salts which can inhibit Taq polymerase activity. Low efficiency real time PCR assay is mainly due to the poor reaction conditions or reagent concentrations which includes sub-optimized concentration of Primers, Probes, Taq polymerase, Magnesium, etc. and the reaction condition includes improper or sub-optimal thermal cycling.

The problem with skewed efficiency in Real time PCR
Efficiencies outside the range of 90–110% may artificially skew results and lead to false conclusions, mainly because targets for comparison will have different efficiencies. In addition, inhibition and poor efficiency can affect assay sensitivity, leading to a smaller dynamic range and decreased versatility.

How to check the efficiency of real time PCR assay is skewed or not???
The best method to determine assay efficiency is to generate a standard curve of template diluted over the range of what will be encountered with the unknown samples and look at the efficiency over that range. It should be as close to 100% as possible. A dissociation curve or gel showing multiple peaks or products means there is a competition for reaction resources that almost certainly will have an effect on the reaction efficiency.

Resolution for Poor Efficiency or Inhibition of a real time PCR assay
Once it is found that the assay is not performing well as 100% efficiency or inhibition is occurring one should take steps to make the assay efficiency to an accepted range, one should strive to achieve an assay efficiency of 100%. The following steps can be used to get the desired assay efficiency.
·       
  • For inhibition, try to remove the highest concentration and analyse the standard curve, this may bring the assay efficiency close to the desired range. This is because of the fact that highest template concentration will also have the highest amount of inhibitors, while diluting inhibitors are also getting diluted to a level which is not inhibitory to the reaction.
  •  Re-purifying the template is another option this can also be done without much difficulty, use enough washing steps to wash off chaotropic salts, which can inhibit the reaction.
  •  Assay optimization is another solution for poor efficiency. It is a laborious process, it can increase the complexity of the assay.
  1. Varying Magnesium concentration can be accessed from a range of 3mM to 6mM which can improve the assay efficiency.
  2. Primer Probe concentration optimization also plays a major role. Primer dimer issues need to be resolved for better efficiency reaction and specificity and sensitivity. Sometimes primer need to be re-designed for better results.
  3. Thermal cycling optimization also plays major role, this needs to be adjusted based on the Tm values of the primers.
Following these should bring the real time pcr assay efficiency to be in the range of 90% - 110%.
Note:  a 100% assay efficiency yield a standard curve slope of -3.32.

Friday, March 22, 2013

Antibody Purification Methods

Antibody Prurification Methods




Antibody purification is a multistep process by which contaminants of source is removed and antibody with high purity is obtained. Antibodies are widely used as injectables and parenteral products for human use. Monoclonal antibodies dominate the antibody field. Before going to the antibody purification process, let’s see the contaminants of the antibody source.



Basic Structure of an Antibody


antibody structure

Antibody is a multi-chain protein, secreted in order to fight with the antigens which enters the body and thereby preventing possible infections. Generally an antibody is a polypeptide of four chains, having two identical light chains and two identical heavy chains. Antibodies are glycoproteins.


Possible contaminants:
Antibodies are produced in mice, rabbits, etc. Nowadays antibodies are expressed in cell culture with good yield. When antibodies are taken from animal source possible contaminants could be serum proteins such as albumin, transferrins and cell degradation products like DNA and cellular proteins. Currently serum free media for cell culture are developed which can ease the antibody purification process. With a combination of chromatographic steps and precipitation methods one can purify the antibody with good yield and resolution.

Purification Methods
The choice of a purification method is based on a these factors:
  1. Nature of antibody,
  2. Nature of feedstock,
  3. Scale of production,
  4. Economics – cost and other factors,
  5. Process Timings, and
  6. Desired purity.
As mention earlier antibody purification is multi step process, which mainly includes:
  • Sample Preparation
  • Capture
  • Initial Purification
  • Secondary Purification
  • Polishing / Formulation
Let’s go in detail on each of these steps for better understanding,



purification and characterization of antibody
Antibody Purification: Step 1: Sample Preparation

Sample prep or sample preparation is the initial step in which crude protein sample is conditioned or making it ready for the initial capture step. Generally this step involves changing pH or Ionic strength, dilution of the crude sample or addition of salts for the ionic strength. These above mentioned techniques may increase the cost in manufacturing so they may not be feasible in the large scale production process. So what is suitable in large scale antibody purification process is to use buffer exchange by size exclusion chromatography or to use ultrafiltration or diafiltration. Dialysis is one common method followed in lab scale level but in production scale it is not feasible.


Crude antibody sample need to be concentrated which is done either by centrifugation or Filtration, sometimes both the methods are combined to get faster results.
Antibody precipitation can be done to precipitate out, salts used for this purpose include ammonium sulphate, Poly ethylene glycol, etc. if the antibody is expressed in cell line media contaminants (dye – phenol red) need to be removed which in-turn can bind to the column and reduce the efficiency of the purification process.


Antibody Purification: Step 2: Capture
Capturing is the first major purification step in a process typically involves binding the antibody to chromatographic matrix, while impurities either flow through or are differentially eluted from the column. The process need to be optimized for better results, good yield and purity.


These are the various chromatographic techniques which are widely used for antibody purification.
  • Immunoaffinity
  • Immobilized Metal Affinity Chromatograhy (IMAC)
  • Ion – Exchange Chromatograhy (IEC)
  • Hydrophobic Interaction Chromatography (HIC)
  • Hydroxyapatite
  • Size – Exclusion chromatography (SEC)
All other techniques, except Hydroxyapatite chromatographic technique, are explained in the previous posts, so let’s look into the Hydroxyapatite chromatography.

In hyroxyapatite chromatography, interaction of the antibody with calcium and phosphate has a major role to play. Mostly elution is done using a phosphate buffer gradient. As this technique has the ability to bind to DNA and separate out idiotypes. Hydroxyapatite chromatography is used mostly in lab scale because only low flow rates can be used in this technique and resin cannot be reused. Due to these drawback hydroxyapatite chromatodraphic application is limited to lab scale purification.

Antibody Purification: Step 3: Secondary Purification
The secondary purification step is selected based on the nature and the optimization requirement of the crude antibody source, the initial capture step can often give purities in the range of 80 to 95%.  However, for higher purity grades in excess of 99% secondary purification is required. In addition to protein contaminants, other impurities such as DNA, endotoxins, viruses, and aggregates need to be removed. In such cases, a multistep procedure is almost inevitable. All the same techniques used for initial capture can be used for secondary purification. Indeed, many of the techniques, such as HIC and Hydroxyapatite chromatography, are used more often used as polishing steps than as initial capture steps.


Antibody Purification: Step 4:  Polishing / Formulation
Final polishing / formulation step can be considered as a part of purification in which it removes conditions that would impair the stability or utility of the antibody in its intended use. As the downstream processing rule, more no of steps results in more loss of the product. Final formulation may be as simple as a straightforward sterilization by membrane filtration through a sterile filter with pores 0.2 lm or less. Another relatively simple formulation step is adjusting the antibody concentration, either by dilution with buffer or by concentration on ultra filtration. In other circumstances, the buffer composition may need to be changed to achieve optimal stability of the antibody. For this purpose, SEC or diafiltration is widely used. A more complex formulation step would be the addition of excipients to confer stability. Finally, the antibody solution may need to be lyophilized/ Freeze dried to confer stability, and the liquid formulation may be changed to be compatible with the lyophilization process.
All the above mentioned steps need to be optimized to have a better yield, resolution and better stability.

Monday, March 18, 2013

Gene Therapy & Ethical Issues

What is gene therapy?
Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including:

• Replacing a mutated gene that causes disease with a healthy copy of the gene.
• Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
• Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently only being tested for the treatment of diseases that have no other cures.

How does gene therapy work?
Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.
A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can’t cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.
The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient’s cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein.
Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body.

Is gene therapy safe?
Gene therapy is under study to determine whether it could be used to treat disease. Current research is evaluating the safety of gene therapy; future studies will test whether it is an effective treatment option. Several studies have already shown that this approach can have very serious health risks, such as toxicity, inflammation, and cancer. Because the techniques are relatively new, some of the risks may be unpredictable; however, medical researchers, institutions, and regulatory agencies are working to ensure that gene therapy research is as safe as possible.
Comprehensive federal laws, regulations, and guidelines help protect people who participate in research studies (called clinical trials).

What are the ethical issues surrounding gene therapy?
Because gene therapy involves making changes to the body’s set of basic instructions, it raises many unique ethical concerns. The ethical questions surrounding gene therapy include:

• How can “good” and “bad” uses of gene therapy be distinguished?
• Who decides which traits are normal and which constitute a disability or disorder?
• Will the high costs of gene therapy make it available only to the wealthy?
• Could the widespread use of gene therapy make society less accepting of people who are different?
• Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

Current gene therapy research has focused on treating individuals by targeting the therapy to body cells such as bone marrow or blood cells. This type of gene therapy cannot be passed on to a person’s children. Gene therapy could be targeted to egg and sperm cells (germ cells), however, which would allow the inserted gene to be passed on to future generations. This approach is known as germline gene therapy.

The idea of germline gene therapy is controversial. While it could spare future generations in a family from having a particular genetic disorder, it might affect the development of a fetus in unexpected ways or have long-term side effects that are not yet known. Because people who would be affected by germline gene therapy are not yet born, they can’t choose whether to have the treatment.

Saturday, March 16, 2013

Radial Flow Chromatography (RFC) vs Axial Flow Chromatography (AFC)

Biotechnological processes are advancing day by day and in production scale the volume is going higher and higher, recombinant protein purification involves two or three downstream purification steps. Radial flow chromatography was first introduced for gas – solid catalytic reactions in packed bed columns.
Radial flow chromatography is used as an alternative to the axial flow chromatography which was used for preparative and large scale purification processes. In radial flow chromatography, mobile phase enters radially and enters the centre rather than axially as in the axial flow chromatography. The mobile phase enters through outside tube and merges into the center tube. As compared to the axial flow chromatography, radial flow chromatography column provides larger flow area and shorter path length. It allows higher volumetric flow rate with lower pressure.


working of radial flow chromatography


Problems of Axial Flow chromatography in scale up
Axial flow chromatography has limitation when volumes are high, especially axial flow chromatography becomes troublesome when considering for scale up processes, one of the main drawback is, in order to accommodate larger volumes increasing the column size too much is impractical. When column size is increased excessive pressure drop will be there and by resulting in compromising process resolution and yield.

Pros and Cons of Radial Flow Chromatography
  • Radial Flow Chromatography provides shorter flow path with higher cross sectional area, this has same effect as pancake like axial flow chromatographic columns.
  • RFC columns requires less floor space as compared to AFC columns.
  • Flow distribution problems are there in axial flow chromatographic columns and Radial flow chromatographic columns.
  • Due to the short flow path of RFC columns pressure drop will be minimal and can handle higher volumetric flow rates in RFC columns as compared to the AFC columns.
  • Since pressure drop is minimal, bed compression problems won’t be there even with higher flow rates.
  • RFC is good choice for affinity chromatographic separations.
  • Scale up in RFC is easy because increasing height doesn’t have much effect of flow distortion problems.
  • The main problem with RFC is the limited resolution because of its short flow path.
  • Increase in flow path in larger extent leads to flow distortion problems.
  • RFC has no use in analytical HPLC because of the limited resolution.
  • RFC is not suitable for size exclusion chromatography, since SEC depend on the flow path for the resolution.

Purification of Plasmid DNA using PEG Precipitation

Minipreparations of plasmid DNA can be used as a templates in dideoxy sequencing reactions whose products can be analysed in automated machines. The length of the sequence established on run using the machines largely depends on the purity of plasmid minpreps, the addition of one or two steps in the procedure of plasmid miniprep by Alkaline Lysis by SDS method can increase the purity of plasmid by large fold, which will result in reproducible results with sequence length of more than 600bp on the final sequence run product.

Procedure for PEG Precipitation
  1. To 50 µL of miniprep plasmid add 8.0µL of 4M NaCl and 40µL of 13% PEG 8000 (w/v), incubate on ice for 20 – 30 mints.
  2. Centrifuge at maximum speed for 10mins at 4oC, collect the pellet by carefully removing the supernatant by gentle aspiration. 
  3. Rinse the pellet using 500µL of 70% Ethanol. The colour of the pellet changes to milky white at this stage. 
  4. Carefully remove the supernatant and rinse the pellet again with 500µL of 70% ethanol. 
  5. Remove the ethanol and keep the tube in inverted position till the trace off ethanol evaporates from the pellet. 
  6. Dissolve the pellet in 20 – 30 µL of sterile water. 
  7. Analyse the purity and integrity of the plasmid on agarose gel before submitting for sequencing. 
  8.  Plasmid with high purity and good quality is ready.

Reverse Phase chromatography – Methods and Principle

Reverse Phase chromatography separates proteins and peptides based on the difference in hydrophobicity, Reversible interaction with the hydrophobic chromatographic matrix/medium results in the separation. As the sample is loaded, Binding to the chromatographic matrix takes place, altering the conditions results in elution of the protein or peptide from the matrix. The reverse phase nature of column matrix causes strong binding, usually harsh conditions (organic solvents or ion pairing agents) are required to elute the protein out from the matrix.
<! --adsense-- > In Reverse Phase high purity can be achieved. Since harsh conditions are used proteins and enzyme with final tertiary structure need to be maintained may not suitable for purification using RPC since the protein or enzyme activity may lose during the process.
RPC used is often used in the final purification of oligonucleotides, peptides and proteins. It is ideal for analytical separations such as peptide mapping.

Reverse Phase HPLC separation: Overview


Reverse Phase HPLC separation


Select suitable Hydrophobic Ligand
Ligand is the material on which proteins will interact with and separate based on the hydrophobicity. Commonly used ligands in RPC are C4, C8, and C18 n-alkyl hydrophobic ligands. Select suitable ones based on their degree of hydrophobicity required for the separation.
Highly hydrophobic molecules bind tightly to strong hydrophobic ligand like C18.
Suitable matrix which gives high resolution and purity need to be selected, screening of RPC media can be done to achieve this, trying from low hydrophobicity to high hydrophobicity screening is usually done.

Sample volume and binding capacity
RPC is a binding technique which is independent on sample volume. Total binding capacity depends on the matrix used and the conditions provided for the binding. Matrix should be selected in such a way that one should get high resolution with high sample loading volume.

Sample and column Preparation
As like any other column chromatographic separation process, sample should be free of particulates, this can be achieved by filtering the protein sample with no or low binding filters. Sample with particles can lead to clogging and results in poor resolution and or damage to the system due to back pressure generation and other factors.
Columns of RPC need to be equilibrated with enough column volumes of equilibration buffer so as to give perfect binding condition for the proteins.

Storage
Column need to be stored as per the specifications of the column supplier.

Tuesday, March 5, 2013

Yeast Culture - cell number, Cell Size - Methods and Calculations

Growth and culturing yeast 

Humans have cultured the yeast S. cerevisiae for thousands of years. It is at or near the top of the “first domesticated organism” list. Beer and wine are considered by many to be the pinnacle of human achievement. The ancients in their wisdom fermented sugary foods they could gather as a means to preserve the nutritional value of fruits. The additional recreational value in the consumption of such fermented preparations did not go unnoticed. Small amounts of fermented material were used to inoculate foods in order to obtain reliable preparations.

Yeast can be grown on agar plates or in liquid using defined or complex media.






Feeding Yeast

Yeast can be grown in many kinds of media, in liquid cultures or on agar surfaces. Wild yeast need only a carbon source, ammonium as a nitrogen source and a few vitamins and off they go. Yeast can grow on many different carbon sources from sugars such as glucose (aka dextrose, which they like to ferment to ethanol), galactose, sucrose, raffinose, to other carbon compounds like glycerol and ethanol. Our lab strains havemutations that render them unable to grow on minimal medium (YMD, the Y stands for Yeast, the M for minimal and the D for dextrose). In such cases we may add nutrients they need and cannot make from scratch, for example the base uracil or the amino acid leucine. This method is called the "drop in" method where a supplement is added to minimal medium, eg YMD + leu. So the leucine requiring-strain will not grow on YMD but will on YMD + leu.

Some researchers use an alternative method in which a complete medium with everything even the most debilitated yeast strain needs is present in a mix of purified vitamins, nutrients and minerals. This is called "synthetic complete medium" (SC) and when it has dextrose it is called SCD. To identify a leucine requiring mutant we would "drop out" the leucine and make SCD – leu. In this case the leucine requiring strain would not grow on the SCD – leu, but would on SCD. I know this seems confusing, but we will do both in this class, it is good for budding yeast geneticists (ie geneticist of budding yeasts, and budding geneticists) to learn both ways. Really they are very much the same, but one needs to know what is in or missing from the medium to know the phenotype. So this extra challenge will serve to keep us all on our toes.

Rich media are also called undefined media or complex media. These do not contain known amounts of nutrients but are extracts of different things that are rich and contain all the goodies in various forms to support growth. They are not good for defining auxotrophic phenotypes because most auxotrophs grow readily on rich media.

Objective: Setting up cultures

Tools- sterile petridish, toothpicks, loop, YPD plates, yeast culture (bottle of red tail ale or a yeast culture provided)

Directions: Open culture (5-10 ml).

(If using beer with live yeast, leave bottom 10 ml and decant the rest in a glass. Save decanted beer (to drink later). Swirl bottom of bottle and pour into sterile Petri dish).

Streaking a plate: Flame the loop until it is glowing red. Let it cool in the air. Touch the side of the agar to make sure it is cool (hissing and spitting indicate that it is still hot). Take a loopful of liquid, or just a touch of a colony from the culture to be grown. Patch this in a small area of the agar plate near the side. Flame the loop again and let it cool. Streak through the patch of cells and out onto the agar surface. Repeat each time streaking through the last streak, in order to “dilute” the streak. The idea is to obtain isolated colonies that have arisen from a single cell.

For simple transfers, a sterile toothpick can be used to pick up a small amount of cells and make a small patch on a new plate.

Setting up a liquid culture: From a plate, take a sterile loop and pick up cells from a colony. Disperse into the liquid.

From a liquid culture; make sure the cells are well suspended. Measure a known volume using a sterile pipette (or with a sterile-tipped pipetter). If you wish to start the culture at a known cell concentration, you will need to know the cell number of the innoculum.

Yeast will grow at different rates depending on the temperature. Yeast will grow relatively quickly at temperatures from 37C down to 18C and more slowly at temperatures down to 5C.

Most Saccharomyces cannot survive temperatures in excess of 42C.

How to mate yeast:

Mix two strains of yeast on a plate on which they can both grow (YPD). Incubate at least 6-8 hours, better overnight or 24 hours. Patch or streak to selective medium on which only the diploid and not either haploid, can grow. Test that the strain is diploid by confirming that it sporulates. Multiple crosses can be done simultaneously by crossreplica plating.



YP-Media/ Plates

            Ingredients                                                                 Amount

            Yeast extract                                                                10 g
            Peptone                                                                       20 g
            Bacto-Agar                                                                   20 g
           
            Water                                                                          900 ml

Place a magnetic stir bar before autoclaving.
2          Separately sterilize
            Glucose                                                                       20 g
            Water                                                                          100 ml
Autoclave solutions 1 and 2 separately Mix the two thoroughly and then pour plates.

YPD liquid media is made as above except that solution 1 does not contain Agar. Store solution 1 (liquid media) in 90 ml aliquots and solution 2 in 100 ml aliquots.

YM Plates/media

            Ingredients                                                                 Amount
           YM Media
1          Yeast Nitrogen Base                                                      6.7 g
            (without amino acids)
            Bacto-Agar                                                                  20 g
            Water                                                                         900 ml
2          Separately sterilize
            Glucose                                                                      20 g
            Water                                                                         100 ml

Autoclave solutions 1 and 2 separately and Mix the two thoroughly and then pour plates.


Use of a microscope (Bright field and phase contrast)

Our microscopes can be used for either conventional bright field or phase contrast microscopy. If a specimen is pigmented or stained, use the bright field adjustment. If a specimen is unstained, as most of our yeast preparations will be, use a phase contrast adjustment. Phase contrast is a way to get image contrast using the differing refractive indexes of the different materials in the biological sample. It uses constructive and destructive interference to make different parts of the sample look brighter or darker, respectively.

To make this work, the scope needs to be properly adjusted! YOU must learn (or perhaps re-learn) your way around the microscope. It isn't that hard, and unless you are a brute and force parts that obviously won't move, you won't break it. Two simple rules are "don't force it" and "keep your mitts off the glass parts" (use lens paper).

To use the microscope, please pay close attention to the following steps:

Illumination: A small unit called a transformer converts the 120 volt from the standard AC outlet into smaller voltages (2.5V, 3.5V, 4.5V, 5.5V and 8V) depending on how you insert the plug into the transformer. See the diagram.

The different voltages correspond to illumination brightness- the higher the voltage the brighter the light in the microscope. As you change from low to high power (10x to 100x), you may need to use brighter light. Therefore use the lower voltages for the lower magnification objectives (10x) and use the higher voltages for the 100x objectives.

To start out, insert the microscope plug into one of the lower voltages (on the transformer) since you will be using low magnifications first. Then plug the transformer into the main power outlet and turn the switch on- the lamp should go on.

Carefully prepared wet mounts will be a aid to you as you familiarize yourself with this instrument. Slides and coverslips should be clean. An air bubble under the coverslip will make it easy to focus on the specimen plane. Excess liquid should be blotted with a wipe.

Begin observing cells with the 10x objective and increase magnification as needed.

Typical sequence of action for alignment is:

Clamp slide onto microscope stage and position slide using the mechanical stage knobs so that the specimen appears to be in the optical path

Focus objective on an air bubble or specimen.


Use a culture of E. coli and a culture of yeast

Yeast cell number determination: How many yeast do I have??

Goals

1. Learn to determine yeast cell number by at least two methods:

Petrof-Hauser counting chamber

Optical density at 600 nm (light scattering)

2. Determine over what range of optical density measurements is cell number proportional to optical density at 600nm

3. Determine cell number represented by OD600=1

Objective: count yeast cells in culture. Determine the relationship between cell number and OD600

We will use two different methods of determining cell number: optical density at 600 nm and direct microscopic cell counts using counting chambers.

We will compare these methods and calibrate them to each other.

Stationary cultures of yeast will be available. Medium for diluting yeast to different cell concentrations will also be available.

Method1- optical density measurements:

1 Turn on the spectrophotometer and set the wavelength to 600 nm

2 Using the stationary culture, make the following dilutions: 1:5. Then use the 1:5 dilution and further dilute it two-fold each time till you obtain a 1:160 dilution.

3 Zero the spectrophotometer using the dilution medium (diluent) and the undiluted culture as well.

4 Measure the OD of each dilution and note the values.

Method 2 (Optional): Viable cell count

After measuring the OD of the culture, plate an appropriate volume of diluted cell suspension so that 50-300 cells are spread on the plate. Use the following factor to estimate this: 1 OD = 3x107 cells/ml

To be sure you are in the right range, plate 5 times more and 5 times less than your best estimate.

After the plates dry, incubate at 30C for 2-3 days. Count the colonies

Method3- Petrov-Hauser counting chamber

1 Place the cover slip on the chambers (the long way) so that it covers as much of both of the mirrored chambers as possible.

2 charge the chamber with a minimum volume of cell suspension derived from the dilution series of the culture (usually 10-12 ul) by carefully placing the pippetteman tip in the groove just below the coverslip and pushing the fluid in slowly until the chamber is full (but not more).

3 Place the chamber on the microscope. You should see the grid like this if you are under 320-400X magnification (the 40X objective with either 8X or 10X eyepieces:

The entire area of the grid is laid out like this



4 Count the cells in an appropriate grided area. If there are few cells, count all 25 of the  



squares bounded by triple lines. If there are more cells, count only 5 squares (the center and four corner). If there are many cells, count only the center square. Shoot to count about 100 cells per sample no matter how many squares you are counting (Why?).

5 multiply the cell number by the volume factor to determine cells per milliliter.

The chamber grid is 1 mm by 1 mm and the volume above the grid is 0.1 ul. Don’t forget to multiply if you counted only 1 (X25) or 5 (x5) squares. If you have 137 cells in 25 squares, how many cells per ml do you have in your culture? Remember that a ml is equal to 1 cubed centimeter. The volume you are sampling here is 0.1 cubed mm. Can you figure the conversion factor of this volume to a ml?


Cell depth is 0.1 mm

Volume is 0.1 ul

Ruling pattern is 1/400 sq mm

Rulings cover 9 sq mm. Boundary lines of the Neubauer are the center lines of the groups of three. The central sq mm is rules into 25 groups of 16 small squares, each group separated by triple lines, the middle on of which is the boundary. The ruled surface is 0.1 mm below the coverglass, so that the volume of the 16 small squares is 0.0025 cubic mm.

The number of cells per cubic mm = number of cells counted per sq mm x dilution x 10

The number of cells per ml = number of cells counted per sq mm x dilution x 10,000

1 milliliter (ml) = 1000 cubic millimeters
1 microliter (ul)= 1 cu mm

Cell size determination: How big is yeast??

Look at yeast under the microscope.

There are three standard cultures of yeast- a haploid with mating type "a", another haploid with mating type "" (alpha), and a diploid.

Source
Internet Sources

DNA Quantification and Quality Analysis

After isolation of DNA, quantification and analysis of quality are necessary to ascertain the approximate quantity of DNA obtained and the suitability of DNA sample for further analysis. This is important for many applications including digestion of DNA by restriction enzymes or PCR amplification of target DNA. The most commonly used methodologies for quantifying the amount of nucleic acid in a preparation are: (i) gel electrophoresis; and (ii) spectrophotometric analysis. If the sample amount is less, the former method is usually preferred.

A. Agarose Gel Electrophoresis for DNA Quantification and Quality Analysis


This method of quantification is based on the ethidium bromide fluorescent staining of DNA. Ethidium bromide is a fluorescent dye, which intercalates between the stacked bases. The fluorescent yield of the dye:DNA complex is much greater than the unbound dye. UV irradiation at 254nm is absorbed by the DNA and transmitted to the dye and the bound dye itself absorbs radiation at 302nm and 366nm. This energy is retransmitted at 590nm, the reddish-orange region of the visible spectrum. In case of plant genomic DNA, the nucleic acids are electrophoretically separated on a 0.7-0.8% agarose gel containing ethidium bromide at a final concentration of 0.5 ug/ml. The quantity of DNA can be estimated by comparing the fluorescent yield of the samples with a series of standards, for instance, lambda DNA at varying known concentrations. This provides a very rapid and sensitive means of estimating the nucleic acid concentration. A large number of samples with as little as 1-5ng of DNA can be quantified. Besides quantification, it also allows provides the advantage of analyzing the quality of the DNA preparation. Native DNA, which migrates as a tight band of high molecular weight (> or = 40 kb), presence of RNA, and degraded/sheared DNA, if any, can be visually identified on the gel.

Procedure

Prepare a 0.8% agarose gel.

Add 1 ul of 6X gel loading dye to 2-3 ul of each DNA sample before loading the wells of the gel. Addition of dye allows us to note the extent to which the samples might have migrated during electrophoresis, so that it can be halted at an appropriate stage.

Load at least 1 or 2 wells with uncut, good quality lamba DNA or any previously quantified DNA samples (50ng and 100ng) as molecular weight standards.

Run the submarine electrophoretic gel at 70V till the dye has migrated one-third of the distance in the gel.

DNA can be visualized using a UV transilluminator and quantified in comparison with the fluorescent yield of the standards.

Note: For SSR and RAPD analysis, it is more important to have good quality DNA samples (unsheared/undegraded DNA), than high quantities of DNA. In contrast, RFLP analysis requires larger quantities of DNA, since the technique is not PCR-based.

B. Spectrophotometric Determination

Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of the concentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm) if the DNA sample is pure without significant contamination from proteins or organic solvents. The ratio of OD260/OD280 should be determined to assess the purity of the sample. This method is however limited by the quantity of DNA and the purity of the preparation. Accurate analysis of the DNA preparation may be impeded by the presence of impurities in the sample or if the amount of DNA is too little. In the estimation of total genomic DNA, for example, the presence of RNA, sheared DNA etc. could interfere with the accurate estimation of total high molecular weight genomic DNA.

Procedure

Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260nm as well as 280nm.

Add 10 ul of each DNA sample to 900ul TE (Tris-EDTA buffer) and mix well.

Use TE buffer as a blank in the other cuvette of the spectrophotometer.

Note the OD260 and OD280 values on spectrophotometer.

Calculate the OD260/OD280 ratio.

Comments:

A ratio between 1.8-2.0 denotes that the absorption in the UV range is due to nucleic acids.

A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers.

A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or phenol. In either case (<1.8 or >2.0) it is advisable to re-precipitate the DNA.

The amount of DNA can be quantified using the formula:

DNA concentration (ug/ml) = (OD260 x 100 (dilution factor) x 50 ug/ml ) / 1000

Spectrophotomteric Conversions for Nucleic Acids:

1 A 260 of ds DNA = 50 ug/ml

1 A 260 of ss oligonucleotides = 33 ug/ml

1 A 260 of ss RNA = 40 ug/ml

Reference

Hoisington, D. Khairallah, M. and Gonzalez-de-Leon, D. (1994). Laboratory Protocols: CIMMYT Applied Biotechnology Center. Second Edition, Mexico, D.F.: CIMMYT.

Saturday, March 2, 2013

Easy Method to Clone a PCR Product

Cloning PCR product or amplicon generated  from non-proofreading (no 3'-5' Exonuclease activity) Taq DNA polymerase is fast method and it has got several advantages.
Bacteria containing plasmids can be frozen, providing a ready supply of amplified material. Because of the variety of available plasmids with different promoters and selectable markers, cloning is also useful when mutations are to be introduced into the fragment before expression, or when sequence tags encoded in the vector are to be added in-frame. The ease with which nucleotide sequences can be added to the ends of PCR products has led to the development of a variety of cloning strategies.

TA Cloning exploits the terminal transferase activity of  DNA polymerases such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product.

TA Cloning Plasmid


This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. The PCR products with dA overhang, are mixed with this vector in high proportion. The complementary overhangs of "T" vector and PCR product will be ligated under the action of T4 DNA ligase.
By keeping a final extension step in the PCR run can add up A overhangs at the 3' end.

General Process Flow of cloning PCR Product
  1. Set up PCR reaction (Include Final Extension step - 72 dc for 5mins.
  2. Analyse the pcr product on agarose gel.
  3. Gel purify the product (Gel Extraction).
  4. Quantify the gel extracted product (useful for maintaining insert to vector ratio).
  5. Choose 3:1 ratio of insert (varies based on kit).
  6. Ligate the gel extracted PCR product to the vector(Linear plasmid have 5' T overhangs).
  7. Proceed with Transformation into E.coli. (Make sure you have competent cells ready)
  8. Plate 150 ~ 200 uL on LB Agar having suitable antibiotic and other screening agents (X-gal) depending on the vector used.
  9. Incubate the plates overnight at 37dC.
  10. Screen the clones either by colony PCR or by checking the amplification of the insert.
  11. Select Positive clones and proceed with Glycerol Stock Preparation.
  12. Store Glycerol stocks at -80dC(Long term storage) or -20dC.
Various kits are available for cloning PCR product.
Few of them are

InsTA cloning Kit - Thermo Scientific
Qiagen PCR cloning Kit
PCR Cloning Kit -Invitrogen

Notes:
Gel Extraction of the PCR Product will help increase higher yield and higher transformation efficiency.

Reference
CSH Protocols
Internet Sources

Friday, March 1, 2013

Notes for Successful Chromatography

Sample Preparation for chromatography
Most commonly used technique to clarify the lysate is to centrifuge it at high speed.Protein solution can be filtered to remove particulates from the solution which otherwise block the flow in the column.Filters used should be low protein binding membranes like the PVDF and PES.Loss of protein yield can occur if nylon filters are used. 0.45 µm or 0.2 µm filters are generally used for filtering if there is restriction in the flow through the filters 0.8 µm filters can be used.
While preparing sample for ion exchange chromatography make sure the conductivity is below 10 mS/cm, which will ensure optimal binding.
Proper protein storage methods after purification can increase the stability of proteins.
  • Short term (24 hr or less): 4°C
  • Long term: -80°C. Include 5–50% glycerol, albumin (10 mg/ml), and/or reducing agents like DTT or BME in the storage buffer. Freeze protein in small aliquots in microcentrifuge tubes.
  • Flash freeze tubes using liquid nitrogen, or dry ice/ethanol bath
  • Proetin can also be stored as ammonium sulfate precipitate at 4°C
Column / System Check
Operating pressure is important factor which can influence the chromatographic resolution.Make sure the column is run at the correct pressure. Operating pressure will be mention in the chromatography media manual.
Height equivalent theoretical plate (HETP) count and asymmetry factor (As) are dependable measures of column performance. Note these parameters before first column use to enable
monitoring over time. Always make sure to take your column out of line or put your valves in column bypass prior to purging.
Replace worn frits and O-rings in manually packed columns when they are observed.When attaching a column to your system, you should use the drop-to-drop method to avoid introducing air onto your column. This is a method of filling the column inlet with fluid prior to final attachment.
Occasionally check your UV detector lamp life to ensure it meets recommended output parameters. Tubing on your chromatography system should be short enough to minimize dead volume while allowing component positioning flexibility.Buffer lines should be cleaned with purified water at the end of the day. This is especially important when working at low temperatures where salts can precipitate.
Degas or vacuum filter buffers prior to use to minimize air and particulates in the mobile phase.Occasionally inspect buffer bottles/bags for signs of bacterial growth. Solutions that appear cloudy upon shaking or stirring should be discarded. Treatment with a bacteriostatic agent such as 0.02% sodium azide will prolong solution storage.
Sample inlet lines and sample loops should periodically be cleaned with 1 N NaOH solution, followed by water or neutralizing buffer, to eliminate sample buildup in the tubing. Periodically replace PEEK tubing that comes in regular contact with sample, especially lines connected toyour fraction collector.
Storing all system lines in fresh 20% ethanol when not in use is an effective way to avoid bacterial growth. It is recommended that this is done at the end of each work day.Injection needles are best used with volumes of 1 ml or less. Larger volumes can be more easily managed with a luer fitting attached to your injection port. Calibrate your pH probe daily for most accurate results. Electrodes should always be stored in their designated storage solution or a neutral buffer. 20% EtOH should not be used, as it changes the concentration of KCl in the probe.
Pre-equilibrate the selected column prior to running and observe UV/conductivity behavior stability. Many automated systems allow you to advance to sample injection once complete equilibration is observed.Periodically check system tubing for cracks and bends.
Tubing bends can restrict flow and increase system backpressure.For best results, occasionally sanitize your entire system with 1 N NaOH to clear the flow path from pump to fraction collector.
Detectors
Random spikes in a UV trace that coincide with negative conductivity spikes most often indicate presence of air. Recurring instances of these spikes can often be corrected by purging your system with 1 N NaOH.Signs of pulsation in your gradient may indicate that you are not using the correct size of mixer for your flow rate.S-shaped conductivity gradient curves often indicate a mismatch between mixer size and flow rate.Sawtooth UV signals can be an indication of air trapped in your flow cell. You can often chase the bubble out of your system with 1 N NaOH.
Loading lysate through sample pumps is preferable over using system pumps. The latter increases the chances of sample buildup and contamination in your system.To achieve optimal gradient performance, choose the mixer size that corresponds with the operating flow rate. Proteins and nucleic acids can be monitored together with a multi-wavelength detector. You can also analyze their relative proportions using software for automated chromatography systems.There's a
Know your Protein
Know relevant properties of your protein: pI, molecular weight, hydrophobicity. This will assist in developing a purification strategy. If using IMAC as a capture step, do not add EDTA to your buffers. These will strip the active metals and preclude protein binding.
When creating methods try to minimize redundant steps for clarity of protocol and ease of monitoring. Creating method templates saves future setup time and eliminates the need to monitor entire runs.Tris-based buffers should not be used in IMAC purification, as they will compete the protein of interest for the binding sites.Do not use phosphate buffer systems when usinghydroxyapatite resins, as they will interfere with protein binding. Always name your runs by date or notebook number to facilitate easy data access in the future.
References
Chromatography Success tips and tricks guide for successful preparative chromatography and Protein purification, Biorad
Protein Purification Handbook, GE
Stratagies of Protein Purification,GE


FTA card for DNA Isolation and Storage

FTA Cards are small cards developed to cpature and store DNA from samples.FTA cards are developed by Whatman Ltd, a division of GE group.


FTA card

                                                                                                                     
About FTA card

The FTA card contains certains chemicals that aids in the lysis of cells in the sample ,helps in denaturing proteins and protect nucleic acids from nucleases, oxidative, and UV damage.

Advantages and benefits of FTA card
  • Easy Capturing of nucleic acid in one step.
  • Captured nucleic acid is ready for downstream applications in less than 30 minutes
  • DNA collected on FTA Cards can be preserved for years at room temperature
  • FTA Cards are stored at room temperature before and after sample application, reducing the need for laboratory freezers.
  • Suitable for virtually any cell type.
  • Indicating FTA Cards change color upon sample application to facilitate handling of colorless samples
  • FTA Cards are available in a variety of configurations to meet application requirements

Capture nucleic acids in one easy step

Just apply your sample to the FTA Card. Cell membranes and organelles are lysed and the released nucleic acids are entrapped in the fibers of the matrix. The nucleic acids remain immobilized and are preserved for transport, immediate processing or long-term room temperature storage.

Since captured nucleic acids are preserved, FTA Cards facilitates sample collection from remote locations and transport to labs possible.

FTA cards are available in white and pink. The coloured FTA Cards (Pink) change from pink to white when sample is applied hence it is useful for virtually any tpe of sample:

Blood
Cultured cells
Buccal cells
Plasmids
Solid tissue

                      

Captured nucleic acid is ready for downstream applications in less than 30 minutes
Captured nucleic acids are ready for purification when you are. Just take a punch from the FTA Card, wash with FTA Purification Reagent and rinse with TE-1 (10mM Tris-HCI, 0.1 mM EDTA, pH 8) buffer. DNA on the washed punch is ready to use in applications such as PCR, SNP analysis, and real-time PCR. Since PCR products remain in solution, the punch can be used for multiple amplifications.

Store nucleic acids at room temperature for years
Genomic DNA stored on FTA Cards at room temperature for over 17 years (and counting) has been successfully amplified by PCR. RNA, being chemically less stable than DNA, is best analyzed upon return of samples to the laboratory. Frozen storage is helpful for RNA preservation.

References:
Whatman.com
ehow.com
wikipedia