Alcohol Precipitation

 Introduction

    Last week we worked with the Left Flank, as well as the Kanamycin resistant gene from e.coli, in our effort to transform Deinococcus Radiodurans. This week we worked with the Right Flank. First, we started with making our 1% gel to run the samples through. While we waited for the gel to solidify, we Nano-Dropped our Right Flank sample. 

    From this, we got: 280-2.71, 260-5.10, 230-2.27 

    Finally, our gel had become as solid as we wanted. We loaded our 20 micro-liter sample (2 micro-liters of dye, 18 micro-liters of DNA) into 2 wells, and made sure to load the ladder. After we ran the gel it was time to place it over UV light and carefully remove the fragment. 


This photo is from last week but added to emphasize the procedure. 

    After we extracted the fragments we placed the Right Flank as well as our other fragments into the -20 degrees Celsius freezer to chill overnight. The following morning we began the procedure of Alcohol Precipitation. We added 2 times the volume of each sample. All the samples were around 50 micro-liters, so we added 100 micro-liters of ice-cold ethanol to each. Then placed back into the -20 degrees Celcius freezer for an additional hour. After the hour had finished they went directly to the centrifuge for 30 minutes. After the centrifuge had run its course, we discarded the supernate. Unfortunately for us, the DNA did not pellet as we hoped it would. 

    
    This is a picture of the Eppendorf tube we were hoping would pellet the DNA. I think the tubes might have been too big to pellet anything. Not visible in the picture, but there was a very fine line on the bottom of the tub, possibly our pellet. But it was too fine for us to know. 

    After we tried centrifuging one more time, we were ready to see if we successfully extracted the DNA. 


    Nothing significant, just a picture of all the samples together. 

    After the centrifuging had finished we brought our 6 samples to the Nano-Drop to see if there was any trace of DNA. 



    After we used the Nano-Drop we came to find out that we did not extract the right concentration of DNA. Ideally, we were aiming for 180 ng/micro-liter, but the closest we got was one of the Kanamycin samples being 101.6 ng/micro-liter, and the other being 34.5. 

    Next week we will be conducting the same experiment, hopefully this time with better results. 











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