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Thursday, February 28, 2013

Transformation efficiency (CFU/mg):

Test for Transformation Efficiency
  • Use a stock of pUC19 solution (0.01 mg/ml) to determine transformation efficiency.
  • Thaw or keep competent cells on ice.
  • Add 5 ml (50 pg) control DNA to one polypropylene tube (Falcon 2059) containing 100 ml competent cells. Move the piptette through the cells while dispensing. Gently tap the tube to mix.
  • Incubate cells on ice for 30 minutes.
  • Heat-shock cells 45 seconds in a 42oC water bath; do not shake.
  • Place on ice for 2 minutes.
  • Add 0.9 ml of room temperature LB media (no antibiotic).
  • Shake at 225 rpm 37oC for one hour.
  • Dilute the reaction to 1:400 with LB media (no antibiotic).
  • Plate out 100 ml of the above 1:400 dilution on an LB plate containing 100 mg/ml ampicillin.
  • Incubate the plate overnight at 37oC.
Tranformation Plate

Determining transformation efficiency (CFU/mg):

Transformation efficiency is the efficiency at which bacterial cells can take up the external DNA and express the gene encoded by it.Transformation technique is used in the recombinant DNA technology inorder to transfer the desired genes / foreign DNA into the bacterial cells for expression.

Transformation efficiency can be calculated from the following equation:

(CFU on control plate/ pg pUC19 DNA used)  *  ((1 x 10^6 pg)/ug) *   Dilution Factor   =   CFU/mg
CFU is the colony forming units, pUC19 is a vector which is most commonly used in recombinant DNA technology, examples of other vectors include pBR322, pET, etc.

Tuesday, February 26, 2013

Bacterial Growth Calculation

Bacterial growth is the process of division of one cell to give rise to two daughter cells through binary fission.
Bacterial cells in a batch culture goes through four phases:

  1. Log Phase
  2. Lag / Exponential Phase
  3. Stationary Phase
  4. Death Phase
Bacterial growth curve graph
Bacterial Growth Curve - Log No of Cells Vs Time

Bio-Resource Bacterial growth can be calculated using the simple formula

2^number of generations * initial number of bacteria = total no. of bacteria present after n generations

after each generation cycle one bacteria divides into two, so to calculate no of bacterial cells after n no. of generation just use 2^n no. of generations. Multiplying by the no. of bacterial cells you have inoculated total no of bacterial cells after n no of generations can be calculated.

Example: Bacillus cereus divides every 30 minutes. You have inoculated a culture with exactly 100 bacterial cells. After 3 hours, how many bacteria are present?

In this case you need to calculate no of generations or the divisions bacterial cells takes place in 3 hrs.

3 hours = 3 *60 mins = 180 mins.

TIme for 1 generation is 30 mins,

so in 180 mins bacterial cells divide 6 times (180/30 = 6).

no of generations n =6.

So 2^n ; 2^6 = 64 or 2x2x2x2x2x2

Initial bacterial cells is 100, so

100 x 64 = 6,400 cells

Using the same example, let's say you have determined that your sample contains 6,400
bacterial cells. You know that it incubated 3 hours. How many generations have occurred?

2^n = (log cells at end of incubation ) - (log cells at beginning of incubation)

n= ((log6400) – (log100))/ log2

Therefore, (log 6400) - (log 100) / 0.301 = (3.81 - 2) / 0.301 = 6 generations

To calculate the generation time for a population: 60 min x hours / number of generations

In this example:

60 min x 3 hours / 6 generations = 30 minutes per generation

Practice Problems

1. You perform a serial dilution and determine that the original number of cells in your

sample was 12,000. How many bacteria will be present in 12 hours if the generation time is
15 minutes (assume unlimited food and clean environment)?


12*60 = 720mins

1 generation time = 15 mins

so no of generations in 720 mins = 720/15 = 48

Total no of bacterial cells = 2^n * Initial no of cells

          = 2^48 * 12000
          = 3.4*10^18 cells

2. You determine that a coconut cream pie has 3 million (3 x 10^6 ) Staph. aureus cells in it.
You estimate that the food preparer did not wash his hands and probably inoculated the
cream with 500 Staph. aureus. He also forgot to refrigerate it. If the pie was made 6 hours
ago, how many generations have occurred? How long is each generation?


log (3 x 10^6) - log (500) / 0.301 = (6.47 - 2.7) / 0.301 = 12.5 generations

60 minutes x 6 hours / 12.5 generations = 28.8 minutes per generation

3. Using the generation time from problem 2, how many bacteria would be present after 8
hours at room temperature?


28.8 minutes per generation, 8 hours x 60 min = 480 minutes
total time to grow
480 / 28.8 = 16.7 generations in 8 hours = n


500 x 216.7 = 500 x 1.06 x 10^5

= 5.32 x 10^7 bacterial cells

4. Let's say that flesh eating Strep. pyogenes divides every 10 minutes at body temperature. You fall down and scrape your knee and get infected with 5 Strep. pyogenes cells. After 4 hours, without medical intervention, how many bacteria will be ravaging your body? Let's say that for every 1 million bacteria, a centimeter of flesh is consumed. After 4hours, how much tissue would be lost? Are you still alive and would you want to be? (This problem is not fact based.)


6 generations per hour, 24 generations in 4 hours

5 x 224 = 8.4 x 10^7 bacterial cells

8.4 x 10^7 / 1 x 10^6 = 84 million cells

1 cm of flesh per million = 84 cm of flesh (33.5 inches)

You would be alive but you would lose the leg. We are assuming the infection does not become systemic.


Internet Sources

Saturday, February 23, 2013

Analytical Chemistry Basics

Most of the scientists and chemists consider analytical method as tool for their research or work and are primarily interested in the results they can provide. all are concerned about the accuracy, precision and reproducibility and  reliability of the methods as well as the sources of any errors that can be introduced.One should always seek for ways to improve them so as to exact the very best results (results as close to true values as possible).

Accuracy & Precision
Accuracy of a determination is defined as the concordance between result (observed value) or the true or most probable value (IE, accepted correct value).

The precision of determination is defined as the concordance of a series of measurements of the same quantity.

"Accuracy expresses the correctness of a measurement while precision of a set of results expresses the reproducibility of  a measurement."

Accuracy is always followed by precision, but high degree of precision doesn't imply accuracy.

True Value & Error
Error is at least a small deviation from the true value. True value of a physical measurement can never be known with absolute certainity. only the most probable value can be arrived at. a better value may be arrived subsequently from better techniques.

Expression of errors
Errors may be expressed as absolute errors or relative errors.
The absolute error is the measure of difference between observed value and true value or most probable value of the quantity measured.

absolute error E, = X(obs) - X(t)

Absolute error is always expressed in the units of measurement.It is the measure of accuracy of an experiment.

Relative Error is the ratio of the error to the true value or the most probable value.

Relative error E(r) = (( X(obs) - X(t))/X(t)) * 100%

Classification of Errors
Errors can be classified into two classes : Determinate Errors and Indeterminate Errors.

  • Determinate  Errors (Systemic Errors)

Determinate errors can be avoided or  whose magnitude can be determined.These errors can be eliminated in an improved repetition of the measurements.

  • Indeterminate  Errors (Accidental Errors or Random Errors)

Indeterminate errors are those which cannot be discovered, avoided or eliminated in a repetition of measurements.

Types of Determinate Errors

  1. Operational and Personal Errors
  2. Instrument and Reagent Errors
  3. Method Errors
  4. Additive or Proportional Errors

Types of Indeterminate Errors

  1. Personal Errors
  2. Instrumental Errors
  3. Condition Errors

Wednesday, February 20, 2013

HLA TYPING - Human Leucocyte Antigen system

HLA - Human Leucocyte Antigen system

HLA forms part of the Major Histocompatibiblity Complex (MHC). It is found on the short arm of chromosome 6. MHC antigens are integral to the normal functioning of the immune response.

Essential role of HLA antigens lies in the control of self recognition and thus defense against micro-organisms and surveillance.

HLA comprises two classes:

HLA Class I

HLA Class II

HLA Class I:

A,B,C most significant (other loci eg E,F,G,H etc are not so important in transplantation). It is Expressed on most nucleated cells.

It is present as soluble form in plasma and are adsorbed onto platelets (some antigens more readily than others)

Erythrocytes will adsorb some Class I antigens viz. Bg blood group system (B7,A28, B57….)

HLA B are most polymorphic system and studies have shown is most significant followed by A and then C. it is a 45Kda glycoprotein comprising of three heavy chain domains, non-covalently associated with beta-2-microglobulin (coded chromosome 15) which plays an important role in the structural support of the heavy chains.

Class I molecules are assembled within the cell and ultimately sit on the cell surface with a section inserted into the lipid bilayer of the cell membrane and a short cytoplasmic tail where they present antigen in the form of peptide to cytotoxic T (CD8+) cells

HLA Class II

It is present in five loci DR, DQ, DP, DM and DO

HLA DR, DQ, DP most significant

Expressed on B lymphocytes, activated T lymphocytes, macrophages, endothelial cells ie immune competent cells.

Comprises of 2 chains encoded by HLA genes, alpha and beta each with 2 domains. Hypervariable region is in the beta 1 domain.

HLA Class II present peptide in the cleft to helper T (CD4+) cells. Thus Class II presentation involves the helper-function of setting up a general immune reaction involving cytokine, cellular and humoral defence.

The role of Class II in initiating a general immune response is why they only need to be present on immunologically active cells.


Serology used to be the 'gold' standard. Now being superseded by molecular techniques as they become more robust and time efficient

Cellular rarely used now. Originally used for Class II typing, Molecular Methods are fast and becoming the method of choice. Many laboratories test of choice.


Complement Dependent Cytotoxicity (CDC)

Viable peripheral blood lymphocytes are obtained by discontinous density gradient centrifugation using Ficoll / Tryosil or Ficoll / Sodium Metrizoate at a density of 1.077 at 19º - 22ºC.

Microlymphocytotoxic test: 3 stages

Microlymphocyototoxic test

1.Viable lymphocytes are incubated with HLA specific antibodies. If the specific antigen is present on the cell the antibody is bound.

2.Rabbit serum as a source of complement is added, incubate. If antibody is bound to the HLA antigen on the cell surface it activates the complement which damages the cell membrane making it permeable to vital stains.

Microlymphocyototoxic test 2

3.Results are visualised by adding dye usually a fluorochrome eg Ethidium Bromide although both Trypan Blue and Eosin have been used in the past.

If the reaction has taken place the EB enters the cell and binds to the DNA.

For ease double staining is normally used. We use a cocktail of Ethidium Bromide and Acridine Orange, quenched using Bovine Haemoglobin to allow simultaneous visualisation of both living and dead cells.

Microlymphocytotoxicity test 3

Test is left for 10 minutes and then read using an inverted fluorescient microscope.

A mixture of T and B lymphocytes can be used for HLA Class I typing.

B lymphocytes are required for HLA Class II typing by serology. (Normal population 85-90% T and 10-15% B cells)

This can be achieved using a number of methods.

Microlymphocytotoxicity test 4

In the past neuraminidase treated sheep red blood cell rosetting and nylon wool have been used.

Immunomagnetic bead separation is the current method of choice.

It utilises polystyrene microspheres with a magnetisable core coated in monoclonal antibody for a HLA Class II b chain monomorphic epitope. Positive selection.

Pros and cons


Easily performed does not require expensive equipment.

Takes around three hours to perform.

Low level resolution, with good antisera reliable results.


Requires large volumes of blood.

Requires viable lymphocytes.

Difficult to find good antisera for rarer antigens in different populations

Cellular typing

Not / Rarely used by laboratories these days.

Requires panels of homozygous typing cells.

Cell culture method therefore takes a long time. Labour intensive involves use of radioisotopes.

Molecular Typing

All commonly used molecular methods require good quality genomic DNA. There are numerous methods for extraction of DNA from whole blood.

There are 'in house' methods based on Miller et al's Salting Out which are cheap and easy but labour intensive.

There are also numreous commercial kits available such as individual matrix capture columns, beads and semi automated systems. This however can increase the cost per extraction from around 65p to £3.60p.

All methods rely on DNA extraction from the nucleated cells following cell lysis and protein digestion.

The application of molecular techniques to HLA typing began around 1987 when the Southern Blot technique was used to identify restriction fragment length polymorphisms (RFLP's) associated with known serological DR/DQ and cellular Dw defined specifities.

Around 1992 polymerase chain reaction (PCR) methods were developed.

Most methods currently used have a PCR element within the technique.


Three steps per cycle– denaturation, annealing and extension. Amplification is exponential yielding 2 power n where n = number of cycles.

The introduction of the programmable Thermal Cycler revolutionised the use of PCR within the routine laboratory.

PCT SSP (Sequence Specific Priming)

Can be used for HLA Class I and II typing using a panel of primer pairs either for low to medium resolution whereby primers amplify groups of alleles or high resolution whereby primer pairs amplify specific alleles. Each PCR reaction takes place in a separate tube therefore the number of tubes depends on the level of resolution. Each tube also contains a pair of primers for part of the human growth hormone gene as an internal control. These are at a much lower concentration thus do not compete with specific primers.


Electrophoresis is used following amplification. PCR product is run out on an agarose gel containing ethidium bromide. Each product moves according to its size and is compared to a molecular weight marker.

Interpretation: every tube should produce an identical sized product as internal control and either a specific band or not dependent on whether the allele(s) is/are present or not.

Results are visualised using 312nm UV transillumination and recorded either by video imaging or polaroid photograghy.

PCR SSOP ( Sequence Specific Oligonucleotide Probes)

'Dot blot' in house method usually whereby one labels ones own probes with Digoxigenin

'Reverse dot blot' normally commercial where specific oligonucleotide probes are attached to a nylon membrane. Dynal and Innotrans for example produce such kits.

Amplification: DNA of interest is amplified by a single pair of biotinylated primers which flank the whole of exon eg exon 2 of the HLA DRB1 gene. PCR amplifies all the alleles in the exon.

Hybridisation: PCR product is denatured and then added to a 'well' containing the nylon membrane with the bound probes and incubated with hybridisation buffer . PCR product hybridises to probes with complementary sequences.

Excess product is washed away during a series of wash steps.

Temperature is VERY important during these stages.

Visualisation of results is achieved by incubating with a conjugate and enzyme often streptavidin and horse radish peroxidase which binds to the biotin of the PCR product and then adding a substrate. Band with PCR product turn blue.

Strips will have internal control bands to show the test has worked.

Interpretation is usually achieved by entering the band pattern into a computer programme.

This is an excellent method for low resolution batch testing.

Can be semi automated.

Pros and Cons


Does not require viable cells

Samples do not have to arrive in the lab the day they are taken

PCR SSOP good for batch testing

Can be semi automated


Requires good quality DNA

Require a degree of redundancy within the primers used

Sequence of alleles must be known.

Sequence Based Typing (SBT)

DNA sequencing is the determination of the sequence of a gene and thus is the highest resolution possible. Sequence based typing involves PCR amplification of the gene of interest eg HLA DRB1 followed by determination of the base sequence. The sequence is then compared with a database of DRB1 gene sequences to find comparable sequences and assign alleles. This method also allows for detection on new alleles.

Other molecular methods:

Reference Strand Conformational Analysis (RSCA) Offers sequence level typing without the need to sequence. Assigns HLA type on the basis os accurate measurement of conformation ie shape dependent on DNA mobility in polyacrylamide gel electrophoresis (PAGE). Complex and difficult technique not taken up by labs for routine use.

Luminex technology
– SSOP based. Just beginning to be introduced into laboratories for routine use on non urgent samples.


HLA Typing by B Montague

Wednesday, February 13, 2013

Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR

A good note on Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR from applied biosystems.

Get the tutorial

Reference / Source:

Applied Biosystems

Tuesday, February 12, 2013

Primer and Probe Design for Real Time PCR - Quantitative Assays:

Primer and probe designing softwares are available online which can aid in easy designing of primer probe for the real time PCR quantitative assays. When designing only PCR primer pairs, other general primer design programs could be used such as:Primer Quest available free of charge at: and Primer Select by DNAstar.
Designing primers is one of the most important steps of a real-time PCR experiment. Several software packages are available to help with the design includingPrimer3, a free system from MIT . There are several considerations when designing real-time PCR primers. The product should be short, the two primers should have similar Tms (ideally within 0.5oC of each other but no more than 1 oC apart), and the primers should have low or no self complementarity (to avoid primer dimers). In addition, many people try to design primers that span introns or cross intron/exon boundaries. In this way, only cDNA from mRNA gene transcripts will be amplified, not genomic copies of the gene.

Overview of Primer Probes in Realtime PCR 

Primer Probe in real time PCR
                                                             Image Source

The guidelines for designing primers / probes for Quantitative assays:
· select the probe first and design the primers as close as possible to the probe without overlapping it. Amplicons of 50 to 150 bp are strongly recommended. If absolutely necessary product size could be increased up to 200-250bp at most
· keep primer/probe GC content within 30-80%
· avoid runs of identical nucleotides, this is especially true for guanine, where runs of four or more Gs should be avoided
When designing primers:
· Tm should be within 58°C to 60°C
· the last 5 bases at the 3' end should have no more than two G's or C's
· keep the annealing Ts of the primers as close as possible
· select primer pairs with minimal number of potential primer dimers and primer hairpins as possible
When designing probes:
· Tm should be within 68°C to 70°C
· no Gs on the 5' end
· select the strand that gives the probe more C than G bases

Before to proceeding with real time PCR, it is necessary to test the primers on a PCR reaction to ensure that the primers amplify the gene of interest at the right size and that the primers are specific (i.e. no other bands present on the gel except that of the expected size). In doing this, it is important to run your test PCR under conditions as close as possible to those for real time PCR . Also, the best results are achieved with magnesium concentration at 3mM and with optimal concentration of Taq.
Efficiency of the real time PCR quantitative assay

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); 
click here to know how to generate a standard curve for a real time PCR quantitative assay

where m is the slope;
b is the intercept;

slope (m) = - 3.2746

Amplification efficiency E = 10^(-1/-3.2746) = 2.0

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 % or >105%, one should modify the assay by redesigning your primers and probes.


PCR Application guide, Bio-Rad

Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR, Applied Biosystem

Mathematics in molecular biology and Biotechnology

Technical Resource, RRC Core Genomics Facility, University of Illinois at Chicago

Internet Sources

Monday, February 11, 2013

Real time qPCR vs Digital PCR

Detection of low levels of pathogen

Low level detection of pathogens can be done using qPCR which will produce clear presence/absence results with high-throughput and low PCR can provide unambiguous result.

Viral reference standards

Relative quantification is done using nucleic acid standards or controls by qPCR. Unavailability of standards makes it difficult. Digital PCR is unique among viral detection and quantification methods because it can directly quantify DNA or cDNA prepared from RNA—thus, digital PCR can be used to create reference standards or controls for use in other assays.

Single cell analysis

Real-time PCR enables high-throughput, inexpensive screening of many single cells, but generally requires pre-amplification of samples. Digital PCR, however, enables sensitive, absolute, and precise measurement of single-cell gene expression, without pre-amplification of samples.

Copy number variation

qPCR with TaqMan® Assays and digital PCR can produce unambiguous results for copy number variation detection applications. However, digital PCR offers a more precise approach for discriminating between larger numbers of copies per genome.


Real time PCR Handbook, Invitrogen
Real time PCR Application guide, Biorad