Urban Farming Grows Up

Urban Farming’s mission is to create an abundance of food for people in need by planting, supporting and encouraging the establishment of gardens on unused land and space while increasing diversity, raising awareness for health and wellness, inspiring and educating youth, adults and seniors to create an economically sustainable system to uplift communities around the globe.

INDIRECT ELISA

INDIRECT ELISA :

INDIRECT ELISA The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies).

INDIRECT ELISA :

INDIRECT ELISA Advantages of indirect detection Wide variety of labeled secondary antibodies are available commercially. Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Immunoreactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. 

DIRECT ELISA

 

DIRECT ELISA :

DIRECT ELISA The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point.

DIRECT ELISA :

DIRECT ELISA Advantages of Direct Detection Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated. Disadvantages of Direct Detection Immunoreactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another. Little signal amplification.

 

COMPETITIVE ELISA

COMPETITIVE ELISA :
COMPETITIVE ELISA In this Unlabeled antibody is incubated in the presence of its antigen. These bound antibody/antigen complexes are then added to an antigen coated well. The plate is washed unbound antibody is removed. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.

ELISA : Enzyme-Linked ImmunoSorbent Assay

How the Test is Performed

Blood is typically drawn from a vein, usually from the inside of the elbow or the back of the hand. The site is cleaned with germ-killing medicine (antiseptic). The health care provider wraps an elastic band around the upper arm to apply pressure to the area and make the vein swell with blood.
Next, the health care provider gently inserts a needle into the vein. The blood collects into an airtight vial or tube attached to the needle. The elastic band is removed from your arm.
Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding.
In infants or young children, a sharp tool called a lancet may be used to puncture the skin and make it bleed. The blood collects into a small glass tube called a pipette, or onto a slide or test strip. A bandage may be placed over the area if there is any bleeding.
The sample is sent to a laboratory where the targeted antibody (or antigen) is linked to an enzyme. If the target substance is in the sample, the test solution turns a different color.

How to Prepare for the Test

No special preparation is needed.

How the Test Will Feel

When the needle is inserted to draw blood, some people feel moderate pain, while others feel only a prick or stinging sensation. Afterward, there may be some throbbing.

Why the Test is Performed

This test is often used to see if you have been exposed to viruses or other substances that cause infection. It is often used to screen for current or past infections.

Normal Results

Normal values depend on the type of substance being identified. Normal value ranges may vary slightly among different laboratories. Talk to your doctor about the meaning of your specific test results.

What Abnormal Results Mean

Abnormal values depend on the type of substance being identified. In some people, a positive result may be normal.

Risks

Veins and arteries vary in size from one patient to another and from one side of the body to the other. Obtaining a blood sample from some people may be more difficult than from others.
Other risks associated with having blood drawn are slight but may include:

• Excessive bleeding
• Fainting or feeling light-headed
• Hematoma (blood accumulating under the skin)
• Infection (a slight risk any time the skin is broken)

Alternative Names

Enzyme-linked immunoassay; EIA

References

Ashihara Y, Kasahara Y, Nakamura RM. Immunoassay and immunochemistry. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods. 21st ed. Philadelphia, Pa: Saunders Elsevier; 2006:chap 43.

Plasmid DNA Extraction Using Alkaline Lysis Method

Plasmids can be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. One commonly used technique developed by Birnboim and Doly involves alkaline lysis. This method essentially relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low-pH potassium acetate. This gives selective precipitation of the bacterial chromosomal DNA and other highmolecular-weight cellular components. The plasmid DNA remains in suspension and is precipitated with isopropanol.
Materials:

• Luria Bertani (LB) broth bacteria culture medium: 1% Tryptone, 0.5% yeast extract, 200 mM NaCl. Sterilize by autoclaving in suitable aliquots. In order to ensure retention of the plasmid, media should be supplemented with the appropriate antibiotic(s).
• 1.5 mL Microfuge tubes.
• Sterile tubes: Must have a volume of at least 10 mL to ensure good aeration.
• Lysis solution: 200 mM NaOH, 1% SDS. Store at room temperature.
• Resuspension solution: 50 mM glucose, 50 mM Tris-HCl, pH 8.0, 10 mM ethylene diamine tetraacetic acid (EDTA). Keep at 4^oC to prevent growth of contaminants.
• Potassium acetate (neutralizing solution): 3 M potassium/5 M acetate. For 100 mL, take 29.4 g of potassium acetate, add water to 88.5 mL, and 11.5 mL of glacial acetic acid. Store at room temperature.
• TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
• Isopropanol.
• 70% Ethanol.

Methods:

• Take a number of separate sterile tubes and place 2 mL of L-broth into them. Inoculate from individual bacterial 37°C overnight with shaking.
• Transfer each culture to a labeled 1.5-mL Eppendorf tube, and centrifuge for 30 s at high speed in the microfuge.
  Here, a tight creamy pellet may be seen.
• Decant the supernatant and place tubes in a rack vertically for 5-10 s. Remove any of the remaining liquid by aspiration with a fine Pasteur pipet.
• Add 100 microliters of resuspension solution into each tube, close the lids, and resuspend the bacteria in each tube by shaking or vortexing to dissociate the bacterial pellet.
• To each tube add 200 microliters of lysis solution and mix by inverting the tube several times.
  The solution should quickly turn transparent and become more viscous indicating bacterial
  lysis has taken place.
• Allow at least 2-3 min for lysis to take place and leave the tubes to stand for 60 s before opening. This will allow the liquid to return to the bottom of the tube.
• To each tube add 150 microliters of neutralizing solution and invert the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
• Centrifuge the tubes for 2-5 min at full speed in a microfuge.
• Place new sterile tubes into a rack, label them, and add 250 ~ of isopropanol to each tube.
• Remove the tubes from the microfuge, being careful not to disturb the precipitate.
• Remove the supernatant with a 1-mL pipet, avoiding the white precipitate as much as possible.
  Some of the precipitate may float, so it is critical to use a pipet and disposable tips to
  recover the supernatant rather than pouring it.
• Transfer the liquid phase into the new set of labeled tubes containing the isopropanol.
• Vortex the tubes for 5-10 s and centrifuge the tubes in the microfuge for 30 s at high speed. The plasmid DNA precipitates as a white pellet.
• Decant the supernatant and wash the pellets by adding 750 mL of 70% ethanol, vortex briefly, and centrifuge at high speed for 30 s.
• Decant the ethanol, and centrifuge again for 10 s to collect the remaining ethanol at the bottom of the tubes. Carefully aspirate the remaining ethanol and leave the tubes to air dry on the bench for 5 min.
• Dispense 50 microliter of TE into each tube, and resuspend the pellet
  It is not advisable to vortex, as this may lead to DNA shearing. The sample is best left for
  3-5 min with occasional finger flicking of the tube.
• Take 10 microliters of the resuspended pellet and analyze by agarose gel electrophoresis (see on my posting entitled Agarose Gel Electrophoresis of Nucleic Acids

Size Exclusion Chromatography

Size Exclusion Chromatography (also known as gel filtration) separates molecules based on molecular size. This chromatography can be applied using resins or membrane. With membranes, the smaller molecules pass through while the larger molecules (above a certain size cut-off) are held above the membrane. With resins, the larger molecules pass/flow through the resin and are collected first while the smaller molecules take longer to flow through because these smaller particles get held up within the pores of the resins. Therefore, with resins, the sample passes through the resin in decreasing molecular weight. Common size exclusion applications include concentration, fractionation, desalting and buffer exchange.  

Size exclusion is one of the easiest chromatography methods to perform because samples are processed using an isocratic elution. In its analytical form, size exclusion can distinguish between molecules (e.g. proteins) with a molecular weight difference of less than a factor of two times. In this application, the porosity of the filtration media to be used is selected to provide high resolution in the molecular weight range of interest.