In my project, the process of collecting information can be traced to four major steps:
The following are brief outlines of the major steps. For a detailed account of the procedures, click to open up a Microsoft word document
Serial Dilutions
- The purpose of making serial dilutions is to change the concentration of the virus or the RNA/DNA to be amplified – this helps us to visualize and understand at which concentration of DNA produces visible product
- Basically, serial dilutions are changes in the concentration of DNA template.
- In my project, I prepared 7 dilutions: 100 through to 10-6.
RNA Extraction
- The purpose of RNA extraction is to extract and purify the RNA that is to be duplicated/amplified. The nucleic acid sample may come from a variety of sources: blood, tissues, archived tissues, frozen tissues, etc. In my case, the RNA came from a prepared stock of avian influenza virus (H5N2).
- The samples are lysed and ethanol is added to provide good/ideal binding conditions. The lysate is loaded onto the silica-gel membrace of the RNeasy column to which the RNA binds. All proteins and other contaminants are washed away with buffers and pure RNA is eluted in water.
- RNA is extracted from the 7 dilutions prepared from an earlier step.
- The following procedure applies to one serial dilution.
Mastermix Preparation
- Preparing a mastermix is a step in the process where the PCR reagents/component parts of PCR are put together before the reaction can take place.
- The reagent volumes are pre-calculated on a formulated excel spreadsheet.
- For each of the 4 different concentrations of MgCl2, I will be having 8 reaction tubes (one tube for each dilution and one for a control) and therefore a total of 4concentrations x 8 tubes = 32 total.
Performing PCR
- The purpose of the real-time PCR is to amplify the RNA that was extracted from the influenza virus and produce numerous copies. After performing the PCR, we can analyze the results for its efficiency by creating a series of graphs.
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