Week 7 2/19-2/25: Caeni and pRad1 transformation

 Introduction

    As our project with MIC comes to an end, my partner and I will transition towards transformation of d.caeni with pRad1. Unlike MIC, transformation is a lot denser and more complicated since it is regarded as a parasexual process that involves two partners: exogenous DNA and a recipient cell. For this transformation, we will be working with pRad 1 as our exogenous DNA and d.caeni as our recipient cell. 

    To get ahold of pRad 1, we must extract it from E.coli which is known to have the pRad 1 plasmid inside. Below is our map and outline of the transformation. 

Procedure


1. Obtain E.coli sample from freezebacks (we want fresh new samples) 

2. Streak and grow E.coli in LB with ampicillin plates (the stock concentration of amp: 25 ug/ml)

3. Grow E.coli in LB with ampicillin broth (the stock concentration of amp: 25 ug/ml) 

    ----> The reason to why E.coli must be grown in LB and amp is because since E.coli shows significant  resistance towards amp as an antibiotic compare to other species, when growing E.coli in amp, we can ensure that the samples are fresh E.coli cells and we are able to contain any contamination. 

4. Do plasmid extraction on E.coli to obtain pRad 1 (we are going to use the Zyppy Plasmid Kit)

5. Load the extracted plasmid into a 1% gel to know if it is pRad 1 

6. Streak and grow d.caeni sample from freezebacks in both R2A plates and TGY plates (we want to try R2A as it is a good alternative media to grow d.caeni in since we were struggling to grow it in TGY) 

7.  Grow d.caeni sample in both R2A broth and TGY broth 

8. Do competent cells of d.caeni 

    ----> This step is crucial to our transformation because it makes the cells competent and efficient enough to have the exogenous DNA mixed in. For our competent cells, we will have d.caeni go under heat stock by using CaCl2. 

9. Do transformation with the competent cells and extracted plasmid. 

    ---->  After letting the cells incubate for 16 hours, we will plan to streak them onto TGY plates with 3 ug/ml of chloramphenicol. Chloramphenicol is another antibiotic that E.coli is resistant of. If cells were to grow onto the plates, it can show to us that we might've successfully transformed d.caeni as it is not resistant towards chloramphenicol. 


Results

 To ensure that the cells that grew on the chloramphenicol plates are simply not just dead cells, there are many ways we can confirm this. 

-We can confirm this by growing colonies in normal TGY broth to see if the cells actively grow. 

- Another method is performing PCR on the cells and loading them into a gel. This can confirm that the cells have the sequence that shows resistance towards chloramphenicol.               

    


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