Day1—7/19/2023
1. PCR expanding: PCR, which refers to polymerase chain reaction, is a fundamental technique used to copy or amplify DNA segments. In this experiment, PCR helps develop three types of elements: GFP, UAS, and SPA, which will be used in the following experiments.
2. Electrophoresis: In this experiment, the tested DNA could be compared with the marker to see whether the expanding result is successful.
3. Media preparation (including the K+ and amp kind, and -Trp -leu and -Trp -leu -His -Ade kind): The configuration of the culture medium is beneficial for the growth of E. coli and yeast, which will be used in further experiments and will help to identify whether the experiment is successful.
Day2~3—7/20/2023~7/21/2023
1. Digestion: It prepares for homologous recombination and uses digestion to let the ring shape become a linear carrier
2. Homologous recombination: This method is used to connect UAS and GFP to BD and SPA1 to AD in this experiment.
3. Electrophoresis: Electrophoresis can make sure that the DNA fragment that has been extracted is working for the DNA specimen.
4. Heat-shock: It is to introduce exogenous DNA molecules (AD and BD plasmids) into the receptor cells (Escherichia coli) so that the receptor cells can obtain new genetic traits and grow and reproduce on a Petri dish, thus forming an ideal colony with the plasmids needed for the experiment.
Day4~5—7/22/2023~7/23/2023
1. Plasmid Isolation: Plasmid isolation is a way to extract plasmid from the cell. This method is used in this experiment to extract plasmid AD from E. coli since there is a shortage of this plasmid in the lab.
2. Construction of a dual enzyme digestion system: None of the E. coli bacteria coated yesterday grew out, so it must be done again today. However, it was found that the enzyme used to make the AD part was used up, so a new one had to be made.
3. homologous recombination: Homologous recombination is a common way to connect a segment to the cloning vector. This experiment uses this method to connect UAS and GFP to BD and SPA1 to AD.
4. Heat shock conversion: To introduce exogenous DNA molecules (AD and BD plasmids) into the receptor cells (DH5α E. coli) so that the receptor cells can obtain new genetic traits and grow and reproduce on the culture dish, thus forming an ideal colony with the plasmids needed for the experiment. (Because the E. coli planted on the plate yesterday did not successfully develop, it’s necessary to repeat the process and try to form the ideal colony with the plasmids needed for the experiment).
|
Sample number |
concentration |
purity |
|
1 |
1.317 |
1.83 |
|
2 |
0.775 |
1.86 |
|
3 |
0.694 |
1.79 |
|
4 |
1.513 |
1.78 |
|
5 |
1.738 |
1.80 |
Day6—7/24/2023
1. Colony PCR is used to check if the two plasmid systems BD-CRY2-GFP and AD-SPA1 have been successfully constructed. The last try to develop E. coli was successful. For further development in the experiment, it is necessary to preserve strain and save it for later use.
2. Colony PCR validation: To test whether BD, GFP, AD, and SPA1 have successfully linked. Whether BD and AD exist can be seen by whether E. coli had grown on the K+ and amp culture dishes.
3. Electrophoresis: In this experiment, whether BD and AD are constructed with GFP and SPA1 is shown as whether DNA strands have appeared.
4. Validation plasmid: It is to verify whether the plasmid in E. coli is the target plasmid (BD-CRY2-GFP and AD-SPA1)
5. Rocking incubation: Rocking incubation is commonly used to accelerate the incubation time. In this experiment, the method is used to incubate E. coli.
Day7—7/25/2023
1. Plasmid isolation: It is to extract plasmid BD-CRY2-GFP and AD-SPA1. It’s necessary to remove the use-acquired DNA to continue doing yeast two-hybrid.
2. Yeast two-hybrid: BD-CRY2-GFP and AD-SPA1 protein can be transferred into the competent cell and successfully get to the yeast two-hybrid stage.
3. Flasks Culture: Coat the yeast from the yeast two-hybrid system on a 2-deficient plate for cultivation, ensuring that the shaken colonies contain plasmids BD-CRY2-GFP and AD-SPA1 in their bodies.
Two days off
7/26/2023~7/27/2023
Day8—7/28/2023
1. Plasmid isolation: Extract the plasmid of BD-CRY2-GFP and AD-CIB1 from E. coli to go on to the yeast two-hybrid process.
2. Yeast two-hybrid: BD-CRY2-GFP and AD-SPA1(experimental group), and BD-CRY2 and AD-CIB1(control group) plasmids can be transferred into the competent cell, and therefore successfully get to the yeast two-hybrid stage. Then, through coating the yeast from the yeast two-hybrid system on a 2-deficient plate for cultivation, ensuring that the shaken colonies contain plasmids both BD-CRY2-GFP and AD-SPA1(experimental group) and BD-CRY2 and AD-CIB1(control group) plasmids in their bodies.
3. Plasmid Extraction: Preparation of yeast two-hybrid.
4. Yeast culturing.
Day9—7/29/2023
1. Wiki documentation and lab work review with the instructor.
Day10—7/30/2023
1. Carry out blue light and darkness control test and prepare for test.
2. In the meantime, we also set up different volume concentration of yeast to compare the sensitivity of two yeast under blue light conditions.
Day11~12—8/1/2023~8/2/2023
1. We observe the yeast growth against different volume concentration and easily noticed that at low concentrations of yeast, CRY2/SPA1 yeast continues to grow compared to CRY2/CIBI yeast, which essentially stops growing.
2. Another group of experiments for blue light and dark condition comparison also drew the conclusion that the yeast possesses the blue light reactivity.
Day13-8/3/2023
1. Use fluorescence inverse microscope to observe the GFP expression.
2. Use β-gal to test whether the yeast two-hybrid resulted plasmid is active enough.
Day14-8/4/2023
1. Result analysis and Wiki documentation.