E. coli Transformation, Plasmid Miniprep and NanoDrop™ Introduction Bacterial transformation is the insertion of DNA into a competent bacterium, which will result in the plasmid expressing the altered gene. A plasmid Miniprep isolates the plasmid DNA by lysing and neutralizing the DNA, followed by a series of washes to get rid of the debris and finally adding an elution buffer to obtain the DNA. A NanoDrop™ determines the absorbance of a DNA sample at 260 nm and its purity at a ratio of ~1.8. The purpose of the experiment is to use competent E. coli cells to transform them into E. coli cells resistant to Ampicillin, and to perform a plasmid Miniprep to isolate the DNA and use the NanoDrop™ to assess its purity and concentration. Methods …show more content…
An isolated colony from the pCas9-GFP Ampicillin plate was transferred on a sterile tip to a tube containing 5 ml of LB Ampicillin, then it was incubated at 37°C/255 rpm. Glycerol stock. 500 μl of 30-50% Glycerol and 500 μl of overnight cultures were added to an Eppendorf tube and stored at -80°C for future use. Zyppy™ Plasmid Miniprep. A bacterial cell pellet was obtained from a 600 μl bacterial culture grown in LB medium through centrifugation. Then 600 μl of water, 100 μl of 7X Lysis Buffer, and 350 μl of cold Neutralization Buffer were added to the cell pellet. 700 μl of supernatant was obtained and it was transferred to a Zymo-Spin™ IIN column. After it was centrifuged, 200 μl of Endo Wash Buffer and 400μl of Zippy Wash Buffer were added to the column. The Zymo-Spin™ IIN column containing the DNA was placed into a sterile collection tube and 30 μl of Zippy Elution Buffer was added to obtain the plasmid DNA. Thermo Scientific NanoDrop™ 1000 Spectrophotometer. 2 μl of Zippy Elution Buffer and 2 μl of plasmid DNA were added to the NanoDrop™ plate to measure its concentration. Results In figure 1, the incubation of pCas9-GFP resulted in multiple colonies throughout the LB Ampicillin …show more content…
pCas9-GFP cultures in LB Ampicillin plate. 150 μl of pCas9-GFP that was incubated overnight at 37°C generated many colonies that are visible throughout the antibiotic plate. Once the plasmid DNA was obtained from the Miniprep, the ratio and concentration were assessed using the NanoDrop; the results yielded 2.317 and 146 ng /µL respectively. Table 1. Zippy Elution Buffer and plasmid DNA ratio and concentration. The NanoDrop™ measured the concentration and ratio of 2 µL plasmid DNA along with 2 µL Zippy Elution Buffer. Zippy Elution Buffer Plasmid DNA Well ID SPL4 SPL8 260 0.087 0.146 280 0.025 0.063 260/280 3.48 2.317 ng/µL 87 146 Discussion The growth of pCas9-GFP in the Ampicillin plate (Figure 1) showed that the transformation was successful because pCas9-GFP has the gene needed to be resistant to Ampicillin; therefore, it can thrive in an environment containing the antibiotic. However, as seen in Table 1, the ratio of the plasmid DNA was 2.317, which is higher than the given ratio for a DNA sample. The results from the NanoDrop™ indicated that the plasmid DNA was not pure and it was