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Thursday, May 24, 2012

Download Molecular Workbench (MW)

Download Molecular Workbench (MW)
Today I would like to share a link for a software Molecular Workbench(MW), provides visual, interactive computational experiments for teaching and learning science.
Physics, chemistry, biology, biotechnology, and nanotechnology with interactive simulations can be explored using this.
Molecular Workbench is open source and it can be downloaded Free of Cost.

Wednesday, May 16, 2012

Biosafety Cabinets (BSC), Classes / Types of Biosafety Cabinets & its Advantages


Biosafety cabinet (BSC) also known as Biological Safety cabinets or Microbiological safety cabinets are used to provide primary containment in the laboratory when the investigator is handling a potentially infectious materials.
Biosafety cabinets are used in-order to protect the laboratory worker who is handling the infectious materials and also to protect the environment. Most of the BSCs also provide product protection (maintains sterility of the product). It is not safe for the user to work with pathogenic organism in a laminar-flow chamber.
Difference between Laminar-flow (LAF) and Biosafety Cabinet(BSC)
The major difference between laminar-flow chamber and biosafety cabinet is that air which comes out of the biosafety cabinet is filtered using HEPA filter (High Efficiency Particulate Air Filter), where in laminar-flow air blows out unfiltered so there is no protection for the laboratory worker.


A Biosafety Cabinet

There are three types of biological safety cabinets:
Class I: The Class I biological safety cabinet is an open-front negative pressure cabinet. The exhaust air from the cabinet is filtered by a high-efficiency particulate air (HEPA) filter. The Class I biosafety cabinet will provide personnel and environmental protection, but not product protection.

Biosafety Cabinet Class I

Class II: The Class II vertical laminar-flow biological cabinet is an open-front, ventilated cabinet.This cabinet provides a HEPA-filtered, recirculated mass airflow within the work space. Theexhaust air from the cabinet is also filtered by HEPA filters. Thus, the Class II biosafety cabinetwill provide personnel, environment and product protection. While HEPA filters are effectivefor trapping particulates and infectious agents, these filters will not capture volatile chemicals or gases.
Class III: The Class III cabinet is a totally enclosed ventilated cabinet of gas-tight construction.Operations within the Class III cabinet are conducted through attached rubber gloves. When in use, the Class III cabinet is maintained through negative air pressure of at least 0.5 inches water gauge. Supply air is drawn into the cabinet through HEPA filters. The cabinet exhaust air is filtered by two HEPA filters, installed in series, before discharge outside of the facility. The exhaust fan for the Class III cabinet is generally separate from the exhaust fans of the facility's ventilation system.
The use of a Class II cabinet in the microbiological laboratory offers the additional capability and advantage of protecting materials contained within it from extraneous airborne contaminants.
This capability is provided by the HEPA-filtered, recirculated mass airflow within the workspace.Personnel protection provided by Class I and Class II cabinets is dependent on the inward airflow. Since the face velocities are similar, they generally provide an equivalent level of personnel protection. The use of these cabinets alone, however, is not appropriate for containment of highest-risk infectious agents because aerosols may accidentally escape through the open front. When Class III cabinets are required, all procedures involving infectious agents (usually Classes 3, 4 or 5) are performed within them.
The Class II cabinet is the most versatile and economical one available on the market.It is suitable for the containment of biohazardous materials and unlike the Class I biosafety cabinet, it is also suitable as a sterile environment for cell cultures.
A laminar flow clean bench will not provide personnel protection since the air is not HEPA-filtered prior to exhaust across the work area. A laminar flow clean bench MUST NOT BE USED for any work with Class 2 or 3 agents.


Class II, type A: This does not have to be vented, which makes it suitable for use in laboratory rooms which cannot be ducted. This cabinet is acceptable for use of low to moderate risk agents in the absence of volatile toxic chemicals and volatile radionuclides.

Class II, type B1: This cabinet must be vented, with 30% of the air exhausted from the cabinet while 70% is recirculated back into the room. This cabinet may be used with etiologic agents treated with minute quantities of toxic chemicals and trace amounts of radionuclides required as an adjunct to microbiological studies if work is done in the directly exhausted portion of the cabinet, or if the chemicals or radionuclides will not interfere with the work when recirculated in the downflow air.

Class II, type B2: This cabinet must be totally exhausted, with100% of the air exhausted through a dedicated duct. This cabinet may be used with etiologic agents treated with toxic chemicals and radionuclides required as an adjunct to microbiological studies.

Class II, type B3: This must be vented. 70% of the air is exhausted from the cabinet while 30% is recirculated. This cabinet may be used with etiologic agents treated with minute quantities of toxic chemicals and trace quantities of radionuclides that will not interfere with work if recirculated in the downflow air.


Tuesday, May 8, 2012

Nucleic Acid Quantification - DNA/RNA Quantification and Analysis

Nucleic Acid Quantification

Nucleic acids are quantified to check the concentration and purity of DNA/RNA present in the solution is important to know the concentration and purity of the nucleic acid for the use in further applications like PCR, restriction digestion etc. Spectrophotometric analysis is the most commonly used method of quantifying DNA, agarose gel electrophoresis can also be used to analyse the DNA sample for purity.


Spectrophotometric Mehtod

Nucleic acids absorbs UV light in the wave length of 260nm, a solution containing nucleic acid to be tested is exposed to UV light and the absorbance is measured, the more light it absorbs the more nucleic acid your test solution contains.calculations are made according to the Beer Lambert's Law.

NanoDrop is a UV-Vis Spectrophotometer device from thermo scientific, which can quantify nucleic acid from micro volumes of 0.5µL – 2.0µL. Features of this product involves

Direct, easy measurements in less than 5 seconds – just pipette & wipe

Measures DNA, RNA (A260) and Protein (A280) concentrations and sample purity (260/280 ratio)

Large concentration range (2 ng/µL – 15,000 ng/µL dsDNA) without dilutions.

The latest NanoDrop series are NanoDrop 2000/2000c NanoDrop 8000, NanoDrop Lite, etc

A NanoDrop Device


The 260/280 ratio tells us the purity of the sample analysed, Pure DNA sample gives a 260/280 ratio ~ 1.8 and for pure RNA 260/280 ratio is ~ 2.

Similarly, absorbance at 230 nm is accepted as being the result of other contamination; therefore the ratio of A260/A230 is frequently also calculated.. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. 

Residual chemical contamination from nucleic acids extraction procedures may result an overestimation of the nucleic acid concentration and/or negatively influence downstream analysis. Example spectra for 4 common extraction reagents which, if not properly cleaned up, will affect sample purity. 

Image Source : Nanodrop Technical Literature

Checking Contaminants in the Extracted sample

  • A low 260/230 ratio indicates the contaminants absorbing at 230nm or less.
  • A low 260/280 ratio indicates the contaminants absorbing at 280nm or less.
  • A shift in the wavelength trough is indicative of contaminants absorbing at low wavelengths.
  • The wavelength of the sample peak should be at 260nm if contaminants are present the peak may shift.

Nanodrop peak shift

If the sample is contaminated with proteins or other organic compounds, the 260/280 ratio will vary from the above mentioned values.

Agarose Gel Electrophoresis

gel electrophoresis

It is a separation method used to separate nucleic acids based on their size under the influence of electric current. Since Nucleic acids are negatively charged, on applying electric field they move from cathode to anode. Depending upon the size of the nucleic acid to be analyzed suitable gel concentration can be made which act as a sieve to separate nucleic acids based on their size.

Purity of the sample can be analyzed using this method. Some time contaminating DNA/RNA fragments can be removed using this method. After running the sample on gel the band of interest can be spliced out and gel extraction can be done to purify it.

Thursday, May 3, 2012

Role of Sample load and Sample volume in chromatography

Sample preparation for the chromatographic separation is an important step in the chromatographic purification process. There are certain parameters of the sample to be checked before loading to the chromatographic column. Sample load, sample volume, concentration and viscosity of the samples, these are the major parameters which interfere or influence the resolution.

protein sample filter

Filtering a Protein sample - Image Source

Ion Exchange chromatographic(IEC) separation is based on the charge, for principle of Ion Exchange chromatography click here. In IEC, for binding, ionic strength of the sample should be less and in Hydrophobic Interaction Chromatography binding takes place at higher salt concentration. Both these chromatographic techniques are independent of sample volume.
Binding capacity of the matrix should be checked, and amount of matrix required is calculated according to the quantity of protein to be bound.

This is calculated using the formula,

chromatographic matrix / media volume (L) =

Quantity of protein to be bound (g) * (1 + safety margin/100) / dynamic binding capacity(g/L)

Sample applied to the column should be filtered to remove any particulates, and if the sample is viscous it need to be diluted or else it will cause problem while passing through the stationary phase.
HIC and IEX are independent of sample volume.
In case of gel filtration / size exclusion chromatography sample volume is an important factor, gel filtration chromatography is independent of sample concentration (but not more than 70mg/ml). Sample volumes are expressed as percentage of volume of the packed bed.