GELIFICATION - EXAMPLE

In order to better understand the advantages of gelified reaction mixtures in comparison to standard reaction mixtures, the set-up of a PCR reaction will be described as an example (see our Legal Notice).

A. Setting up of a PCR reaction by traditional methods

  • Step 1. Thawing of reagents (15 min): in order to avoid reagent degradation this process, as well as all further reaction steps until introduction in the thermal cycler, is performed on ice.

  • Step 2. Homogeneisation of thawed reagents by vigorous vortexing (5 min).

  • Step 3. Master-mix set up (10 min): a vial containing a Master Mix is prepared. For Master Mix, primers (at least a pair, forward and reverse), reaction buffer, water, MgCl2, dNTPs and polymerase are added. This step must be carefully performed in order to be sure that all reagents are included and contamination of vials containing the reagents is completely avoided.

  • Step 4. Preparation of reaction vials (10 min): once the Master Mix has been prepared, it is aliquoted into the different reaction vials. Again, extreme care must be taken, so that there are no contaminations arising out of the environment or from adjacent reactions vials that may lead to false positive results.

  • Step 5. DNA addition to the reaction vials (5 min): vials will then be introduced in the thermal cycler in order to proceed to the amplification reaction.

    For performance of these 5 steps, physical separation between work areas is highly recommended as well as unidirectional workflow. This is not always possible for a high number of laboratories, and impossible for in-field analysis.

    B. Setting up of a PCR reaction using ready-to-use gelified reaction mixes

  • Step 1. DNA and water addition (5 min): in contrast to setting up reactions in liquid format, gel format vials contain all necessary reagents, so that they are ready-to-use and the setting up of the reaction only involves the addition of the template DNA and water.

    It is also important to bear in mind that gel format vials are stable at room temperaturte, so no ice is required in any point of the process, and risk of false positive results due to contamination or false negative results due to unintentional non-addition of reaction components to the vials is completely avoided.

    In conclusion, use of gel format reduces the aforementioned protocol for liquid format from steps 5 to 1, avoiding most of the critical points associated with a DNA amplification. Therefore, the use of gel format reagents and kits results in a higher specificity, sensitivity, reliability and efficiency of each and every enzymatic reaction where it is applied, in contrast to uncertainty associated with traditional, liquid formats.

    Legal notice: Some of the applications which are described here may be covered by applicable patents in certain countries. This information should be regarded as scientific/technical information and not as a recommendation or stimulation to practice any procedure or to use any product which violates an existing patent, law or regulation depending on country and/or application.