This week, I resumed testing of samples previously extracted from E. coli bacterium. Both protocols submitted for analysis utilized a 0.3 ml TBS and 10 µl SDS solution to lyse the cells, a 55°C heating and ice cooling method combined with a protein precipitation solution to reduce protein contamination and an isopropanol/ethanol wash to precipitate chromosomal DNA and clean the DNA pellet.The protocols differed in the substances used for protein precipitation; protocol one (P1) utilized proteinase K, and protocol two (P2) utilized a guanadine hydrochloride/sodium acetate solution. They also differed in length of precipitation in isopropanol; P1 was in solution for five minutes while P2 remained in solution @ -20°C for five days.
The samples were placed in an electrophoresis gel for analysis. The gels were created @ a ratio of 20 g agarose/100 ml 1 X TAE buffer ratio, with 4 µl of 10,000x concentration SYBY green DNA stain added to solution post-heating.
The spectrometer was also used to measure ultraviolet absorbance ratios of the four samples. Samples were subjected to 260 nm, 280 nm, and 320 nm consecutively.
Results:
DNA!
All four samples showed banding in the gel; P1 sample two contained the most clearly visible banding. The photo that follows is of the samples. The clarity is a bit off due to the age of the camera used to capture the image.
Calculations from the spectrometer readings to follow on my next blog. Happy learning!
DNA!
All four samples showed banding in the gel; P1 sample two contained the most clearly visible banding. The photo that follows is of the samples. The clarity is a bit off due to the age of the camera used to capture the image.
Calculations from the spectrometer readings to follow on my next blog. Happy learning!
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