Day1 8/8/2023
1. Run PCR to amplify the DNA sequence of ELOVA5
|
DNA |
1 ul |
|
Primer F |
2.5 ul |
|
Primer R |
2.5 ul |
|
Max mix |
25 ul |
|
ddH2O |
19 ul |
Procedure
l 94 degrees Celsius for 5 minutes
l 94 degrees Celsius 30 secs --> 60 degrees Celsius 30 secs --> 72 degrees Celsius 1 min, cycles 35
l 72 degrees Celsius 10 mins --> 4 degrees Celsius storage
2. Sterilization of LB liquid media
a) Place the flask containing LB liquid media into the sterilizer
b) Set the temperature to 121 degrees Celsius for 20 minutes
Procedure in details:
-Prepare for Gel Electrophoresis:
TAE preparation, 2% agarose gel preparation
-Run Gel Electrophoresis
Day2 8/9/2023
1. LB media, Prepare for PCR, Run PCR, Perform DNA purification of genes Fat1 and ELOVL5 yielded from PCR.
- Prepare 100 ml LB liquid media
l 0.5g Yeast Extract into the flask
l 1g Trypton into the flask
l 1g NaCl into the flask
l 100mL ddH2O into the conical flask
2. Sterilization of LB liquid media
a) Place the flask containing LB liquid media into the sterilizer
b) Set the temperature to 121 degrees Celsius for 20 minutes
Procedure in details:
l Gel electrophoresis of Fat1 and ELOVL5 yielded from PCR done on day 1
l Reuse gel from the previous step.
3. Plasmid extract
Procedure in details:
l Digestion of Fat1 gene
l Run gel electrophoresis to verify the presence of digested Fat1 gene
l Cultivation of GFP and pET-28a in Kana+ LB broth
Day3 8/10/2023
1. Run gel electrophoresis to determine Fat1 and ELOVL5 gene fragment size, verifying digestion's success.
2. Prepare LB liquid media and separate DNA fragments from the agarose gel for purification. Perform ligation of pET-28a plasmid and target genes.
3. Sterilization of LB liquid media
a) Place the flask containing LB liquid media into the sterilizer
b) Set the temperature to 121 degrees Celsius for 20 minutes
4. Purification of digested Fat1, ELOVL5, and corresponding plasmids
5. Determine the yield and purity of the digested Fat1, ELOVL5, and corresponding plasmids by comparing the sample’s light absorbance ratio of 260nm and 280nm to that of pure DNA
6. Ligation of the plasmids and target genes (25 microliters)
EVOLA5 and Fat1
7. Cultivate DH5- E.coli on LB plate (petri dish)
Day4 8/11/2023
1. Prepare 300 mL of sterilized liquid LB media for bacterial cultivation.
2. Perform homologous recombination of pET-28a-fat, GFP, and elovl5. Run PCR to replicate recombined plasmids. Transform the plasmids into DH5α-E. coli competent cells. Cultivate E. coli on the petri dish.
3. Test the yield and purity of GFP
4. Homologous recombination and PCR
5. Transformation
6. Cultivation on Petri dishes *5(done next to an alcohol burner)
Day5 8/12/2023
1. Prepare Kana+ LB liquid media and transfer E. coli colonies to the LB liquid media.
2. Perform gel electrophoresis and PCR of plasmids pET-28a-Fat1, pET-28a-ELOVL5, pET-28a-GFP-Fat1-ELOVL5, and digested Not1. Transform the plasmids into DH5α-E. coli cells. Extract and purify the DNA fragments from the gel. Conduct Restriction digestion of NotI.
3. Transform the plasmids into DH5α-E. coli cells using the heat shock method
4. Purify the Not1 digested DNA
Day6 8/13/2023
1. Perform plasmid extraction and purification of pET-28a-Fat1.
2. Process glue recovery, and measure the concentration of DNA. Transform the pET-28a-GFP-Fat1-ELOVL5 plasmids into DH5α-E. coli and BL21(DE3). coli competent cells. Cultivate the transformed competent cells on the LB plates.
3. Cultivate the transformed competent cells on LB plates
Day7 8/14/2023
1. Check the growth of E. coli cells. Perform plasmid transformation. Perform OD test, and extract plasmids.
2. Perform plasmid extraction
3. Prepare gel for the SDS page.
4. Gel at the bottom and the top
Day8 8/15/2023
1. Transfer the bacteria cultures into six tubes to centrifuge. Introducing the crack solution and using ultrasonic broken bacteria. Then centrifuge at 13000 rpm for 30 min, and remove supernatant for further purification.
2. protein purification, retrieve the SDS-page, gel electrophoresis
Day9 8/16/2023
1. Plasmid extract
2. Obtained the plasmid pET28a-Fat1 and sent it to Genscript for protein expression level evaluation.
Day10 9/11/2023-10/7/2023
Functional tests
1. Purified Elovl5 to validate its ability to elongate EPA into DPA
2. GC-FID analysis (Testing conducted through professional testing organization-Institute of Agro-products Processing Science and Technology)