Pglo Plasmase

In the LB (pGLO negative), it is expected to not see any colonies growing. As a result of this experiment, it shown any growth colonies but this only had shown a large number of white, circular colonies that were found across the surface of the agar. In the LB/Amp (pGLO negative), it is expected to see any growth colonies. In the experiment, the resulted was no growth colonies because this has Ampicillin and no pGLO. Now, the LB/Amp (pGLO positive), it is expected to have growth colonies in the agar plate. As a result, it was shown growth colonies in the agar plate because has positive pGLO regardless having Ampicillin. In the other hand, both positive pGLO have the same components but in one plate was added Arabinose. The LB/Amp/Ara (pGLO

The reason why it did not inhabit any change after the experiment was conducted is because it had no plasmids added to support the growth and the ampicillin actually killed the bacteria. The plasmid would have served as the vector to insert the gene into the E. coli. The -pGLO LB plate thrived because there was no ampicillin present. While the +pGLO LB/amp/ara plate ended up glowing under the UV light because the GFP was only able to show with the presence of the arabinose, because it is what activates the operon. The +pglo LB/amp plate contained a few colonies but it did not glow under the UV light due to the absence of the

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

14. Use the same loop and technique to spread the same cell suspension (+) on the LB+AMP agar plates. Dispose of the sterile loop in a beaker of germicide.

There were two tubes used in this process: the tube that contained the primary culture and the tube that contained the nutrient agar where the unknown bacteria would grow. First, the inoculating loop was flamed. After removing the caps of both the test tubes, they were flamed to prevent contamination of the unknown bacteria. The inoculating loop was cooled for a few seconds and was then placed into the test tube containing the bacteria. The inoculating loop with the bacteria was placed into the nutrient agar test tube for cultivation. Before the test tubes were capped, they were flamed once again. Also, isolation of the unknown bacteria had to completed. Nutrient agar was placed in the petri dish, and was left to gel for a few minutes. After the agar gelled, the inoculating loop was used to acquire bacteria and streak the unknown onto the plate for

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

Coli. To be able to activate the GFP gene the presence of the sugar arabinose is required. In order to make the E. Coli grow, the presence of ampicillin, an antibiotic, and pGLO is present, however, if ampicillin is present without pGLO it will stop the growth of the E. Coli because the pGLO would not be present to stop the antibiotic. In the experiment E. Coli was tested with the different proteins and plasmids, testing if the plate would glow or grow. If the E. Coli was tested with ampicillin, pGLO, and arabinose the plate would both glow and grow because of the proteins present, however, if the E. Coli was tested with ampicillin and pGLO the plate would neither glow or grow because the proteins were not

Half of each tube’s contents are poured into a new test tube each respectively after the tubes are incubated for 1 hour. One set of tubes is tested for:

If there are any lab’s instructions that I should know, please describe it to me. I am afraid in making any mistake again.

To begin with, we gathered fifteen test tubes in our test tube rack: three arabinose (A), three glucose (G), three mannitol (M), three raffinose (R), and three nitrate broth (N) tubes. We then labeled each test tube with either A, G, M, R, or N on the top of those tubes. Next we labeled these tubes with our initials, the date, and either Escherichia Coli or our unknown bacterium number. We did this so we would know which tubes to inoculate with E. Coli or our unknown and so we would know which tubes are ours. With that, we began inoculating the tubes with one loopful of E. Coli to one set of tubes in the order N to R to M to G to A. We flamed each tube before and after each inoculation in order to maintain aseptic techniques. This step was repeated two more times with each of our unknown bacteria. Finally, all of the tubes were incubated at thirty degrees Celsius. After a week on incubation, we added six drops of naphthylamine and two drops of sulfanilic acid to E. Coli N tube and our unknown tubes. This was to test for a positive

After each technique was used, there were only results for the streak plate and the spread plate. No results were found for the pour plate. The bacteria can be counted for the streak plate, but not for the spread plate. It was found that using the streak plate technique, it would be easier to count the individual colonies. It was also discovered that doing the serial dilution to make sure that the agar is cooled before adding the bacteria.

You will need 11.5 grams of Nutrient Agar mix, a flask, a stir bar, a hot plate, a polystyrene partitian sterile petri plate, and 500 mls of distilled water. You now have to combine the agar mix and the 500 mls of distilled water and mix together. Put the agar into the 500 mls of distilled water and make sure the stir bar is in the flask. Put this mix on the hot plate and bring to a boil. Make sure the agar mix is dissolved before removing from the hot plate. After pouring into the plates, close the lids on them to prevent condensation. Also important is to use plates within one day of making agar. Make sure to label the plates properly(name, date, swabbing area). What is especially important during swabbing is to use sterile cotton Q-tips and to make sure they are not contaminated. To do this, you should not touch the tip of the swab or put it in contact with anything besides the area being tested. This general rule should also apply to the plates. To swab, after collecting the sample, gently roll the tip of the swab against the agar in a zig- zag type motion, making large patterns. This will help you to achieve better colony growth. Make sure to place the lid back on the plates. After swabbing, place petri plates in 37 degree celsius incubator and let stand for 4 days, then record data. When collecting data, make sure to use qualitative and quantitative

The agar plates will be divided into 3 sections (one for each filter paper discs) using any form or marker. Using sterilized forceps, put the filter paper disc at the center of each area in an agar plate. Apply 20µl of each concentration for 10 agar plates. This includes distilled water (negative control), 100% ethanol and the common antibiotic. Observe for the zone of inhibition within 16 hours and above (Tendencia, 2004). To measure the zones of inhibition, use a ruler and record the diameter in terms of millimeters.

Heat the inoculating loop until it glows red. Let it cool then remove a small amount of culture from the agar surface; touch it several times to the drop of water until it just turns cloudy.

TYE ampicillin glucose agar plates: 15g Agar, 8g NaCl, 10g bacto-tryptone and 5g yeast extract should be dissolved in 800ml water (deionised). This solution should be autoclaved before cooling to 50˚C, and adding 1ml of ampicillin solution as well as 200 ml glucose solution before pouring plates. These can then be stored for up to a month at 4˚C, ensuring to dry in a flow-bench before use.