Question

can anyone paraphrase the following procedure for me please. I prefer to be typed because the handwriting sometimes is not clear

First experiment: Bacterial Transformation and Affinity Purification of Protein:

Transformation procedure:

Bring 2 test tubes and label them +pGLO and –pGLO,

Transfer 250 ul of transformation solution to each tube and then place them in ice.

Pick a single colony of bacteria using a sterile loop from the bacterial plate and immerse the loop in +PGLO tube and start spinning the loop to mix, repeat the same step for –pGLO and place both tubes in ice again.

Then, immerse a new sterile loop in the pGLO DNA plasmid stock tube and withdraw a loopful of the plasmid and immerse it in the +pGLO tube to load it and place the tube in ice again. No pGLO plasmid will be added to the –pGLO sample as labeled.

Incubate the tubes in ice for 10 minutes and take the following labeled plates from the instructor: LB/amp + pGLO, LB/amp/ara + pGLO, LB/amp – pGLO, and LB – pGLO. There will be a total of four plates.

Heat hock will start by transforming both tubes to 42C water bath for 50 second and then returning them to ice again.

Add 250 ul of LB nutrient broth to both tubes and incubate at room temperature for 10 minutes.

Using a new sterile pipet for each plate, transfer 100 ul of the –pGLO solution to the, LB/amp – pGLO and LB – pGLO agar plates and 100 ul of +pGLO solution to the LB/amp + pGLO, and LB/amp/ara + pGLO plates.

Spread the suspension using sterile loops and place your plates upside down in the 37C incubator overnight.

Bacterial growth will occur and result can be analyzed the day after.

* For data analysis, observe the result in the plates under normal light conditions and then under UV light and right your observations. This will include observing the number of colonies, colonies color and growth. Transformed bacteria will look green in color under UV with the presence of arabinose sugar in the media and will look white in color if arabinose is absent. No growth will be shown if the bacteria are not transformed.

Second experiment: Affinity purification of green fluorescent protein:

Affinity purification procedure:

Re-observe you plates from previous experiment under natural light and UV light. Identify several green colonies on the LB/amp/ara plate and with the use of a loop scoop up some cells and immerse the loop in a new culture tube labeled (+), the same procedure is done for the LB/amp plate in a tube labeled (-). Disperse the colonies in the culture tubes and shake vigorously. And place in an incubator for 24 hours.

Transfer the entire culture to a 2-milliliter microtube labeled +. Centrifuge for 5 minutes and remove the supernatant.

Re suspend the pellet in 250 ul of TE solution and then add 1 drop of lysozyme and mix then place the tube in the freezer to help rupture the cells.

For bacterial lysis, thaw the microtube and centrifuge for 10 minutes at maximum speed. And prepare the chromatography column.

Before performing the chromatography, add 2 milliliters of Equilibration Buffer to the column, and Drain the buffer from the column until it reaches the 1 milliliter.

After centrifugation, transfer 250 μl of the supernatant into the new microtube and transfer 250 μl of Binding Buffer to the microtube and Place in the refrigerator.

Label 3 collection tubes 1, 2 and 3. Load 250 μl of the supernatant in Binding Buffer into the top of the column and Let the entire volume of supernatant flow into tube 1.

Transfer the column to collection tube 2. Add 250 μl of Wash Buffer and let the entire volume flow into tube 2.

Transfer the column to tube 3. Add 750 μl of TE buffer and let the entire volume flow into tube 3.

Examine all collection tubes using the UV lamp and note any differences in color between the tubes.