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Friday, December 28, 2012

Good Notes on oligonucleotide Yield, Resuspension, and Storage

Oligonucleotides are short stretch of single stranded DNA or RNA molecules used in genetic testing, research and forensic. These are chemically synthesized and has got applications in polymerase chain reaction (PCR),DNA sequencing etc.

Length of oligonucleotide is denoted as '-mer' for example : 18-mer, 22-mer, etc.

Nucleic  acid synthesis columns come in several scales, which are a function of the amount of the 3' nucleoside coupled to the support material. Popular column  sizes Oligonucleotide Synthesis 
range from 40 nmol  to  10umol. Popular support materials  include controlled-pore glass  (CPG) and polystyrene. The CPG columns  are  typically  supplied having pore sizes of either 500 A or 1000 A. The  scale of column used for a synthesis depends on both the quantity and the length of oligonucleotide desired. The greater the amount of 3' nucleoside  attached to the column  support,  the greater the yield of oligo. Polystyrene or large-pore CPG columns  are recommended for oligonucleotides  greater than 50.

oligonucleotides are synthesized at different scales like 0.05umol, 0.2umol, 1umol, etc. depending on the application it is used for.

The number of micromoles of an oligonucleotide synthesized on a column,  therefore, can be calculated by multiplying  the length of the oligonucleotide by the column scale.

A good explanation on calculation of oligonucleotide Yield, Resuspension, and Storage

Integrated DNA Technologies
OligoCalc, Technical guide
Eurofins-MWG Operon,

Metric Prefixes and Scientific Notations

A metric prefix is a shorthand notation used to denote very large or vary small values of a basic unit as an alternative to expressing them as powers of 10. Basic units frequently used in the biological sciences include meters, grams, moles, and liters.Because of their simplicity, metric prefixes have found wide application in molecular biology. The following table lists the most frequently used prefixes and the values they represent. 

Metric Prefix
1 nanogram (ng) is equivalent to 1*10^-9 grams. There are, therefore, 1*10^9 nanograms per gram (the reciprocal of 1*10^-9; 1/1*10^9 = 1*10^-9). Likewise, since one microliter (uL) is equivalent to 1*10^-6 liters, there are 1 x 10^6 uL per liter.

When expressing quantities with metric prefixes, the prefix is usually chosen so that the value can be written as a number greater than 1.0 but less than 1000. For example, it is conventional to express 0.00000005 grams as 50 ng rather than 0.05 ug or 50,000 pg.

Mathematics in molecular biology and biotechnology

Tuesday, December 25, 2012

Pipette calibration : Procedure and Calculations

Under a constant temperature and atmospheric pressure, the density of distilled water is constant. The volume of water can be determined by weighting dispensed water. The calibration of pipette is carried out by gravimetric method. When determining the volume of water, the accuracy of measurements is effected by ambient temperature, atmospheric pressure and relative humidity. These factors are usually combined to give the Z factor, used in calculation of volume of water. Then the calculated volume of water is compared with the theoretical volume to determine the accuracy and precision of the pipette.

Pipette parts
A Micro Pipette

Material and equipment:
  • Ppipette and tips
  • 50 ml beaker and plastic medicine cup
  • Distilled water
  • Temperature meter ( ±0.1℃ )
  • Analytical balance ( ±1.0 mg )
  • Atmospheric pressure meter

procedure pipette calibration
Pipette Calibration

  • Determine the water temperature and record it.
  • Place a beaker filled with distilled water into analytic balance and close the door of balance waiting for equilibrium of inner vapor.
  • Place a plastic medicine cup on the pan and adjust the weight to zero.Put a tip onto the pipette and set the volume which is to be tested.
  • Pre-rinse the tip: aspirate and dispense the setting volume three times and press the push – button on the second stop to remove any remaining liquid.
  • Press the push-button to the first positive stop. Hold the pipette vertically, immerse the tip so that 1-4 mm in the liquid and release the push button slowly and smoothly to aspirate the liquid.
  • Wait one second and withdraw the tip from the liquid.
  • Wipe any droplets away from the outside of the tip using a kimwipe.
  • Place the end of the tip against the inside wall of the plastic cup at an angle of 10-40 °.
  • Press the push-button smoothly to the first stop. Wait one second, change new site and press the push-button on the second stop.
  • Keeping the push-button press to the end, remove the pipette by drawing the tip along the inside surface of the plastic cup and release the push button.
  • Close the door of balance and record the value on the balance display after it has stabilized.
  • Repeat step (6) to (12) 14 times ( the weight display of balance should be adjusted to zero every time)
  •  Eject the tip.
  • Pipette should be calibrated every three months routinely.
Factors Affecting the Accuracy of Air Displacement Pipettes
  • Temperature 
Temperature has many effects on pipetting accuracy. The factor that has the greatest effect 
is the temperature difference between the used delivery device and liquid. The air gap (dead 
air volume) between the liquid surface and the piston experiences thermal expansion effects 
according to the case. This either reduces or increases the liquid amount aspirated into the tip 
along with other effects.
  • Density 
The density (mass/volume ratio) affects the liquid volume that is aspirated into the tip. A smaller dose of liquid with higher density than water is aspirated compared to similar operation 
with water. With lower density liquids the effect is the opposite. This is caused by the flexible 
dead air volume along with the earth gravity. The density of liquids varies also according to the 
temperature. Typically the density for water is 0.998 kg/dm3, for ethanol 0.79 kg/ dm3 and for sulfuric acid (95-98% H 2SO4) 1.84 kg/dm3 (the values apply at temperature of 0 °C).
  • Altitude
The geographic altitude affects the accuracy through air pressure.  The air pressure decreases 
in higher altitudes and the conversion factor Z decreases as well.  Also, with some liquids the 
boiling point decreases quite close to room temperature, which will increase the evaporation 
loss dramatically.
  • Pipetting position 
Pipetting position can also lead to inaccuracy.


Conversion of mass to volume 

V = (w + e) x Z 

V = Volume (µl)  w = Weight (mg) 
e = Evaporation loss (mg) 
Z = Conversion factor for mg/µl conversion

Accuracy (systematic error) 

Accuracy is the difference between the dispensed volume and the selected volume of a pipette.
A = V -V0

A = Accuracy 
V = Mean volume  
V0 = Nominal volume

Accuracy can be expressed as a relative value: 
A% =( 100% x A) / V0

Convert the weight unit of measured value into the volume unit of measured value using the following formula:

Volume ( ml or ul ) = Weight ( mg or ug ) x Z

Z value : conversion factor, which is conversion of density .

Mean Weight = (sample replicate 1 + sample replicate 2 + (etc.))/ Number of Replicates

Mean Volume (Corrected Mean) = Mean Weight × ZFactor

% Inaccuracy = [(Corrected Mean – True Value) ÷ True Value] × 100 

Standard deviation can be expressed as a relative value as cv. 

% CV (Coefficient of Variation) = (Standard Deviation / Corrected Mean) × 100

Calculate the average ( Mean ), accuracy, standard deviation (S.D.) and imprecision ( C.V. ) using the
following formula:

       n              n   - 2

       Σ Xi            Σ (Xi-X)

   -   i=1             i=1      1 / 2

(Mean) X ═ ---------------- ;   S.D. ═(----------------- )

       N              N

N: number of measured value

    S.D.            Measured value

C.V. = ----------- X 100% ; Accuracy = ------------------- X 100%

    Mean            Theoretical value

  • Accuracy value must be 99-101 % and C.V. value must be less than 1 %.
  • Y.C.Chang (Apr. 14, 2007), Laboratory manual of  Clinical  Biochem.  Res.  Lab.                                                                    Dept. of Res. & Edu.Taipei Veterans General Hospital.
  • Roger Schultz, Wisconsin State Laboratory of Hygiene, December 5, 2007
  • Thermo Pipetting Guide

Sunday, December 23, 2012

Estimation of Biomass in Plant Cell Cultures

Biomass is an important parameter which needs constant monitoring to understand the growth process in plant cell and tissue cultures. Cell biomass itself is often the final product, or even if the desired product is a secondary metabolite and the compound is intracellular one needs to have a measure of biomass for several estimations related to the secondary metabolite production.

There are larger no. of methods for estimation of biomass which differ in their degree of accuracy, rapidity, convenience, instrumental requirements etc. some methods may not be suitable for a given culture system.

Measurement of cell number

In very fine suspension cultures cell number may be counted directly in a haemocytometer. Most cultures usually contains aggregates and clump. In such cases cell aggregates need to broken into individual cells prior to counting the number of cells.
  1. Add two volumes of 8% w/v aqueous chromic trioxide solution to one volume of culture suspension.
  2. Heat at 70oC for 2-15 mins.
  3. After cooling, shake the culture mixture vigorously to assist the loosening of cell clusters to individual cells. 
  4. dilute appropriately and count the number of cells under microscope using haemocytometer.
  5. Express the biomass as number of cells per ml of culture after correcting for the dilution factor.
Measurement of Packed Cell Volume (PCV)
  1. Transfer 10ml of culture suspension to a 15ml graduated centrifuge tube. tubes which taper at the bottom are preferable.
  2. Centrifuge for 5 mins at 200gm.
  3. Read pellet volume and express percentage of total volume of the culture suspension.
Measurement of Fresh Weight
  1. Filter the cell suspension through a pre-weighed whatman filter paper on a buchnel funnel under slight vacuum.
  2. wash the cells with distilled water.
  3. Drain fully under vacuum.
  4. Reweigh the cells per filter.
  5. Express the weight of cell biomass as a fresh weight.
Measurement of Dry weight
  1. Filter the suspension through a pre-weighed whatman filter paper on a buchnel funnel under slight vacuum.
  2. wash the cells with distilled water.
  3. Drain fully under vacuum.
  4. Dry cells plus filter paper in a hot air oven at 60oC to a constant weight.
  5. Reweigh the cells per filter.
  6. Express the weight of cell biomass as a fresh weight.

Saturday, December 8, 2012

Generating Standard Curve to analyse the reaction optimization - Real Time qPCR, Calculating PCR Efficiency

Generating Standard Curve to analyse the reaction optimization - Real Time qPCR

A standard curve can be generated using a 10-fold dilution of a template amplified on a real-time system (Example: ABI 7500). Each dilution can assayed in triplicate(its always better to do in duplicates or in triplicates).

Below are the data generated after a Real time PCR run.

Log DNA dilution

The template used for this purpose can be a target with known concentration (e.g., nanograms of genomic DNA or copies of plasmid DNA) or a sample of unknown quantity (e.g., cDNA). Here plasmid is used as the template for the reaction.

The standard curve is constructed by plotting the log of the starting quantity of template (or the dilution factor, for unknown quantities) against the Ct (Cycle Threshold) value obtained during amplification of each dilution.

Below is the standard curve graph generated using the data obtained after the PCR run, here Ct value is plotted against log DNA dilution.

Standard Curve qPCR
Standard Curve qPCR

The equation of the linear regression line, along with Pearson’s correlation coefficient (r) or the coefficient of determination (R2), can then be used to evaluate whether your qPCR assay is optimized.

Real-time quantification (qPCR) is based on the relationship between initial template amount and the Ct value obtained during amplification, an optimal qPCR assay is absolutely essential for accurate and reproducible quantification of the sample.

The hallmarks of an optimized qPCR assay are:

• Linear standard curve (R^2 > 0.980 or r > |–0.990|)
• High amplification efficiency  (90–105%)
• Consistency across replicate reactions.

One should strive to achieve a PCR efficiency above 90%.

qPCR Calculations

As everyone knows in PCR one copy of the template becomes two after the first cycle. the template copy number keeps accumulating as the no of cycles proceeds. it is given by a general formula:

2^n; after 25 cycles one copy of the template becomes 2^25 = 33554432 copies or 3.35*10^7 copies.

If perfect doubling occurs with each amplification cycle, the spacing of the fluorescence curves will be determined by the equation 2^n =dilution factor, where n is the number of cycles between curves at the fluorescence threshold (in other words, the difference between the Ct values of the curves).
for example, with a 10-fold serial dilution of DNA, 2^n = 10. Therefore, n = 3.32, and the Ct values should be separated by 3.32 cycles.

if the PCR assay has 100% efficiency one copy becomes two after one cycle, so how to calculate PCR Efficiency.

Amplification efficiency denoted by E can be calculated from the below equation:

E = 10^(-1/slope);

From the standard Curve chart (y = mx + b)

where m is the slope;
b is the intercept;

slope (m) = - 3.2746

Amplification efficiency E = 10^(-1/-3.2746) = 2.02

Amplification efficiency is also frequently presented as a percentage, that is, the percent of template that was amplified in each cycle.

To convert E into a percentage:

% Efficiency = (E – 1) x 100%

 % Efficiency = (2.0 – 1) x 100% = 100%.

An efficiency close to 100% is the best indicator of a robust, reproducible assay. Low reaction efficiencies < 90% may be caused by poor primer design or by suboptimal reaction conditions. Reaction efficiencies >100% may indicate pipetting error in your serial dilutions or co-amplification of nonspecific products, such as primer-dimers.

When using the method described above to determine amplification efficiency, the presence of inhibitor can also result in an apparent increase in efficiency. This is because samples with the highest concentration of template also have the highest level of inhibitors, which cause a delayed Ct, whereas samples with lower template concentrations have lower levels of inhibitors, so the Ct is minimally delayed. As a result, the absolute value of the slope decreases and the calculated efficiency appears to increase. If the  reaction efficiency is <90 nbsp="" or="">105%, one should modify the assay by redesigning your primers and probes. 


PCR Application guide Bio-Rad
Mathematics in molecular biology and Biotechnology
Internet Sources

Sunday, December 2, 2012

Rapid Diagnostic Tests, Types, Strength and Weakness Applications

Rapid diagnostic tests (RDTs) are diagnostic assays designed for use at the point-of-care (POC), and can be adapted for use in low-resource settings. An RDT is low-cost, simple to operate and read, sensitive, specific, stable at high temperatures, and works in a short period of time. RDTs are already in use for several neglected diseases.


Rapid diagnostic tests (RDTs) are a type of point-of-care diagnostic, meaning that these assays are intended to provide diagnostic results conveniently and immediately to the patient while still at the health facility, screening site, or other health care provider. Receiving diagnosis at the point of care reduces the need for multiple visits to receive diagnostic results, thus improving specificity of diagnosis and the the chances the patient will receive treatment, reducing dependence on presumptive treatment, and reducing the risk that the patient will get sicker before a correct diagnosis is made. Rapid tests are used in a variety of point-of care-settings—from homes to primary care clinics or emergency rooms -- and many require little to no laboratory equipment or medical training.

Rapid Diagnostic Test

RDTs are particularly important in low-resource settings, where:

Harsh environmental conditions combined with limited access to electricity and refrigeration preclude the use of sensitive equipment
Technology, equipment, and training required for more complicated laboratory tests are lacking
Many patients cannot travel easily to the clinic to follow-up on results that take a long time

RDTs can be especially useful with patient samples that can be collected by minimally trained health personal, such as community health workers.1 Body fluids that can be collected non-invasively, such as nasal swabs, urine, saliva, and tears, are preferred as these are most amenable to collection with only minimal training. However, capillary blood collection techniques, such as those used for malaria RDTs, demonstrate that innovation in sample collection can be used to improve the utility of RDTs in low resource settings.

Rather than one specific type of technology, rapid diagnostic tests can be built in a variety of platforms, each with their own benefits and limitations. The vast majority of RDTs in use today for neglected diseases are based on immunoassay technology due to its relative simplicity. These tests generally involve the interaction of a fixed reagent of either target antigen or antibody that is linked to some type of visible detector, that then reacts with a patient sample. Other types of technologies, such as nucleic-acid amplification, may be too expensive and require too much advanced technology to be applicable as a point of care test.

Rapid diagnostic tests have particular value as epidemiological tools, in addition to use as diagnostics. They enable a rapid screening of a potentially affected population, and can be used, as is the case with lymphatic filariasis, as a test of cure to determine when a mass drug administration has been successful. RDTs are less necessary for diseases that are generally accurately diagnosed syndromically, but could prevent over-prescription of antibiotics if used to differentially diagnose fever or diarrhea, respectively.

Common RDT Platforms

There are several different platforms commonly used to build rapid diagnostic tests. The relative utility of common RDT platforms is summarized below.

Lateral flow tests are the simplest type of RDT, requiring only very minimal familiarity with the test and no equipment to perform, since all of the reactants and detectors are included in the test strip. In a lateral flow test, the sample is placed into a sample well and migrates across the zone where the antigen or antibody is immobilized. The results are read after a certain amount of time has passed. Another type of RDT, a flow-through test, obtains results even faster than lateral flow tests, but requires an added wash and buffer step, which can limit its portability and stability.

Rapid Test: Lateral Flow

An agglutination test works very simply by observation of the binding of carrier particles and target analytes into visible clumps, seen either through a microscope or with the naked eye. However, if the binding of the particles is weak, the results of the test can be inconclusive.

Dipstick format RDTs (with binding sites to test for multiple antigens) work by placing the dipstick in a sample. The dipstick is then washed and incubated to prevent non-specific analyte binding. These additional steps can limit their usability in low-resource point of care settings.

Microfluidics, or “labs on a chip” are an emerging area of rapid diagnostic development. Using electrochemical sensors, these tests would include all detectors and reactants in a single portable cassette. For more information, see the Diagnostics Innovation Map Report. The limitations and advantages of each of these test types is summarized in the table below. For more information about the mechanisms of these tests, see PATH’s RDT Info website.

Microfluidics - Rapid Diagnostic Test

Strengths and Weaknesses of RDTs and Common RDT Platforms
The general strengths of RDTs include their ease of use, minimum training requirements, rapid results, and limited need for instrumentation/infrastructure. The general weaknesses of RDTs including their subjective interpretation of readout, low throughput, often limited sensitivity relative to laboratory or reference tests, and need for quality control mechanisms.

RDTs as Non-Neglected Tropical Disease Diagnostics
Rapid diagnostic tests are commercially available for detection of multiple non-neglected diseases and conditions, including pregnancy, blood sugar in diabetic patients, and strep throat. The simplicity of these assays often allows them to be used at home or by minimally trained health care works in both the developed and developing worlds. In the United States, the use of rapid diagnostic tests by doctors is limited as current Medicaid/Medicare reimbursement schemes favor the use the centralized laboratories for diagnosis.

RDTs as Neglected Tropical Disease Diagnostics
One of the particular problems facing the production and implementation of RDTs in low-resource settings is the lack of an evaluative process to determine their real efficacy in the field. For example, the WHO recently issued a policy statement to recommend that rapid serological tests for TB not be useddue to a lack of sensitivity and specificity.4 WHO-TDR and FIND now have lot-testing programs for RDTs for neglected diseases, such as malaria and HIV, to improve standards and recommendations for use of RDTs in the field.

In order to address the ongoing challenge of quality assurance for rapid test, the WHO now independently evaluates RDTs for several diseases, including HIV and malaria, in order to evaluate them for pre-qualification. As with pre-qualification for drugs or vaccines, this designation is the WHO’s method of validating the quality of specific diagnostic assays and manufacturers for those in countries without rigorous and stringent diagnostic approval processes.


P. van Lode (2005). “Point-of-care immunotesting: Approaching the analytical performance of central laboratory methods”. Clinical Biochemistry 38.
Mabey et al. (2004). “Diagnostics for the Developing World.” Nature Reviews Microbiology (2)
WHO News Release (2011). “WHO warns against the use of inaccurate blood tests for active tuberculosis”.
WHO. Malaria Rapid Diagnostic Tests.
FIND. Malaria RDT Product Testing.
WHO. HIV Diagnostic Test Kit.