Pglo Transformation

When placing the large test tube in boiling water, be sure to keep the face of the large test tube away from you or any lab partners with safety tongs.

We first started this experiment by obtaining twelve 15ml test tubes, in which we placed in a rack and labeled each with what

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge tube, since they are getting the exact same

Next prepare the third test tube with an accurate measurement of 5ml sodium carbonate once again labelling appropriately.

Use a test tube holder to put the test tube into a container of boiling water for 5 minutes, or until the solution changes color.

4.Measure 35mL of warm water and add them into each of the 4 test tubes at about roughly the same time. It is essential that the water is warm. Do not seal the test tube.

Use the cotton swab to streak the five labeled nutrient agar plates with the E. Coli K12 bacteria from the tube.

Take a test tube and place it in the test-tube rack. Use a medicine dropper to add 5 drops of water to the test tube. Then add 2 drops of indicator solution to the water.

1st step is to label 3 test tubes A,B,C. 2 step is to put 3 mL of homogenate into test tube A and place in ice water for 30 minutes. 3rd step is to put 3 mL of homogenate into test tube B and set it at room temperature. 4th step is to put 3 mL of homogenate into test C and place in hot bath water. After, add hydrogen peroxide to each test tube and observe what

1 ml of water should be added to the first test tube and make a note. In the second test tube, 1 ml of methyl alcohol should be added. In the third test tube, 1 ml of hexane must be added. Lastly, the fourth test tube will be a control.

A drop of blood is added into each test tube and they are left for 5 min.;

Add 2 drops of soap to the removed test tube, and place it in the test tube holder.

Collect to 2 large beakers both large beakers are to be filled with hot water (labtutor). Then obtain seven conical tubes these will be used to collect the levels of gas, you will also need test tube a stopper and a plastic tube (labtutor). You want to fill the conical tube to at least 50 ml of water (Cressy). Take the four conical tubes filled with water and place two in each beaker, to do this you must invert the tube and cover the release hole as to not lose any water (Cressy). Then place the beakers with the tubes in the bath so they can be at the same temperature as the bath (Cressy). Next mark all of your test tubes in number order to be sure which tube contains what concentrations and pH (Cressy). Having mixed a solution to the specifications of 2.5 ml of glucose in all tubes, 3 ml of yeast in 2 tubes of pH 5, 2 tubes of pH 9, and the single pH 7 tube, the remaining two tubes will contain no yeast as they will be negative controls. Next add 2 ml of pH buffer 3 tubes will receive pH of 5, three will receive a pH of 9 and a single tube of pH 7. Finally add pure water to make sure all test tubes have 10 ml of solution. When making the solutions

7.When water bath is ready, put each test tube into the water bath. Wait 5 minutes.