cDNA Synthesis

    Leading up to the cDNA synthesis other members of the group made and inoculated plates/broth, graham stained and performed RNA isolation and converted RNA to cDNA. 

Image of D.rad Graham Stain
Picture taken through microscope of 
small pink circular cells


    For cDNA, we start with our RNA. After nanodropping the RNA to ensure the concentrations are good enough to perform cDNA synthesis on, we could run a gel to verify the quality of the sample. We than serial dilute the RNA to get our ideal dilution (from previous experiments around 1/1,000 and 1:10,000) 

    We take 1 ng of RNA and combine it Nucleotide free water in order to read 14 μl total volume. We than combine the 14 μl with 2 μl DNase master mix (.5 μl DNase and 1.5 μl DNase buffer). We than use the PCR machine to incubate the reaction. 

DNA Digestion 25°C for 5 min.

DNase inactivation 75°C for 5 min.

than held at 4°C


    After the reaction we add 4 μl of reverse transcription master mix and incubate the reaction again. 

Priming 25°C for 5 min.

Reverse transcription 46°C for 20 min. 

RT inactivation 95°C for 1 min.

than held at 4°C


    The final 20 μl cDNA can't be nanodropped, so the beginning RNA needs to be within the right measurements, and all if the steps need to be as precise as possible with little error. 

    After reviewing the procedure, we went ahead and nanodropped the RNA to ensure it was viable. Unfortunately the RNA decays quickly, and we were unable to proceed with the actual cDNA synthesis. We didn't have enough time to get more RNA, and needed to wait for the next week. With the experiment having a temporary set back, I instead made myself useful round the lab. I filled pipette tip boxes, joined in some conversations about various projects and made various TGY broths and medias. 


RNA nanodrop 


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