Report on preen Plasmid Transform into E. coli D cells:

        preen Plasmid and E.coli D cells had converted carrying the GFP protein. The plates contained the selective antibiotic and it were cultured on it. The transformed E.coli D cells were contained in three microcentrifuge tubes of 1.5mL and were labeled with the numbers in an ascending order. Tube 1 contained the converted cells while the negative, positive, and controls were contained in the tube 2 and tube 3.

        In the aliquots of 50µL each tube, the E.coli D were thawed or broken on ice and contained in three microcentrifuge tubes. pGreen plasmid DNA’s 0.5µL was added in the tube 1. Meanwhile, pUC19 DNA’s 0.5µL was added in the tube labeled three. In the tube 2, only the E.coli D cells were contained. After the addition of LB medium’s 800µL to each of the tube, the incubation and heat shocking of cells occurred at the temperature of 37C in a bath. At 8000 rpm, the cells were centrifuged as well for almost two minutes. Furthermore, each control and transformation was cultured on the plates of ampicillin antibiotic.

Cell Extract’s Preparation:

        The controls and transformed E.coli or pGreen cells’ overnight cultures were gathered from the plated of ampicillin. Into the four sterile microtubes, each culture’s 1mL was transferred. At 12000 rpm, these specific microtubes were centrifuged for almost two minutes. From these microtubes, all the four suspended cells were sent into a new microtube. Meanwhile, the supernatant was eliminated.

        After the resuspending of cell pellet, the remaining cell pellet was operated with lysozyme solution’s 100µL and TE buffer’s 250µL. For twenty minutes, it was incubated in a water bath at the temperature of 37C. The culture was sent to a cycle of freeze thaw for three times. The process was carried out as for ten minutes, the tube was placed in a freezer at the temperature of almost -20C. With the completion of freeze thaw process, the centrifugation of culture was carried out at 13000 rpm for almost ten minutes. Whereas, the supernatant that had the GFP protein was gathered and set on ice.

GFP’s Purification through HIC or Hydrophobic Interaction Chromatography:

        From the transformed E.coli D or pGreen cells, the isolation and purification of GFP was carried out by the performance of HIC. It attached to the column’s sides due to the GFP’s hydrophobicity. This phenomenon gave the access to bacterial proteins to run and pace throughout the column. It isolated the GFP as well.

        The column was cleaned with the Equilibration buffer’s 5mL as it was dived into the waste beaker. Until the buffer’s meniscus had reached the resin bed, it was placed under the column. With the washing of the column, crude extract’s 250µL and binding buffer’s 250µL were mixed and was transferred to the column’s top. Into the four microtubes, 0.5mL’s four elution fractions were collected. It happened after the addition of wash buffer’s 2mL to the column. 0.5mL’s four elution fractions were collected into four more microtubes. It happened after the addition of elution buffer’s 2mL was added to the column.

        Actually, 8 microtubes were filled with the 8 samples. With the 2-3 column volume of wash buffer’s 1.3M and distilled water’s 3-5 column, the HIC matrix was regenerated and the samples were collected.

Determination of GFP’s elution pattern in the cell extract through spectrophotometry:

        At OD 280nm and at OD 490nm, the elution fractions’ eight samples were collected in the HIC’s process. This was carried out for the determination of GFP’s present amount versus the total amount of protein available in the crude extract. In order to measure at the OD 490nm, each sample’s 200µL that was collected in the HIC was sent into a plate of microtiter. They were read as well in order to determine the GFP protein’s presence in the samples. In order to measure at the level of OD 280nm, each sample’s 200µL was sent into the microtubes that were new and properly labeled. The microtubes had elution buffer’s 600µL in them. The samples of amount 800µL in the tubes were sent to the elution cuvettes of UV. For the reading, the Genesis 10 spectrophotometer was used to determine the availability of proteins in the samples.

Determination of Protein through Bradford Assay:

        In order to quantify the presence of all the proteins available in the collected 8 samples at 595nm in HIC, the Bradford assay was utilized. The graphing of BSA standard curve or Bovine Serum Albumin standard curve was carried out for the determination of concentrations of proteins in the purified and GFP and crude extract in HIC.

For the SDS-page Gel Electrophoresis, the preparation of samples:

    The stacking and separating gels were developed by 5% and 12% polyacrylamide. It was processed by the mixing of methylene bisacrylamide and acrylamide in a buffer based on SDS. For the 10X stock solution of 10% SDS stock solution and Tris-Glycine, a 1X TGS’s 470mL buffer (1X Tris-Glycine; 0.1% SDS) was developed.

        125mL TGS buffer was relied on for the filling of 0.5cm inner chamber above the shorter glass plates’ top. The process was carried out at the gel cassettes. Meanwhile, 200mL was added to the mini tank’s lower chamber. In the tubes, the samples of GFP gel and CE gel in the sample buffer of 1X TGS were placed and boiled for almost 2 minutes. For five minutes, the GFP samples and CE samples were cooled down. They were centrifuged and were loaded in gel’s different wells. For almost one hour, the gel was run at energy supply of 200 volts. Each gel was rinsed for five minutes, three times. With the Bio-Safe Coomassie stain, the gel was stained for one hour. With a large amount of water, the gels were destained for the efficient outcomes.