Blog Post #1: The effects of Amyloid Binding dyes on the Cryspovirus in Cryptosporidium Parvum

The purpose of my project is to detect the cryspovirus in Cryptosporidium Parvum. This will be done by cloning GFP, a green fluorescent protein into the virus dsRNA. The GFP gene is inserted into the cryspovirus will make it easier to enumerate the number of virus in Cryptosporidium Parvum.
First I clone out the GFP gene from the pCMV-GFP vector using PCR. I will then extract the PCR product out by the gel extraction technique. From that gel, we will be removing the GFP band containing the restriction sites needed and inserting it into the cryspovirus. To obtain GFP we first need to extract the DNA as well. We will be extracting the DNA from E. coli. The first thing that needs to be done is E.coli with GFP needs to be grown on an agar plate. So the Agar broth and agar were made and Ampicillin was added to both solutions. The agar was poured into an agar plate and left for solidification. Once solidified GFP was streaked onto the plate and left to incubate. The GFP grew very well. Test tubes were made with the agar broth made and tips containing cultures from the E.coli plate. The test tubes have to be incubated while being shaken.  When they grow the liquid will be cloudy. After they grow, QIAprep spin miniprep kit is used to extract the DNA. Once the DNA is extracted it needs to be cleaned by using the DNA clean and concentrator kit. Once our DNA is ready we will PCR it and use it on our gel. We are looking for a specific band and if that band shows up we will remove it from the gel and remove the DNA from the gel. A gel extraction kit is used to remove the DNA. Once the DNA is removed it will be inserted into the cryspovirus dsRNA. I have to extract dsRNA by excystation of Cryptosporidium Parvum oocysts. This breaks the oocysts (shells) that hold the sporozoites. The RNA in the sporozoites is then extracted. The RNA is extracted so we can use PCR to clone in restriction sites. We are currently using different PCR conditions to clone out the different regions of the viral dsRNA.

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