Label one sterile 15 mL tube “+plasmid” and the other tube “-plasmid.” You will also need to label the four agar plates as follows: LB/Amp +plasmid, LB/Amp -plasmid, LB +plasmid, and LB -plasmid. Using a sterile pipette transfer 250 µL of ice-cold calcium chloride to each tube and place both tubes in a beaker filled with ice. Transfer E. coli from the starter plate to the “+plasmid” tube by using a sterile inoculating loop. Spin the loop in the tubes’ solutions to make sure that all of E. coli’s cell mass is completely in the solution. The solution should turn into a milky white color. Afterwards, return the tube to the iced beaker. Repeat the same transfer process for the “-plasmid” tube. Add a loop of plasmid DNA to the “+plasmid” tube only using a clean and sterile inoculating loop and use the same loop to stir the plasmid with the cells. Do not add any plasmid DNA to the “-plasmid” tube. Put the “+plasmid” tube back in the ice and incubate both tubes for about 15 minutes. After 15 minutes, heat shock the …show more content…
Using your finger, gently tap the tubes to mix the Luria broth with the cell suspension. The test tubes will need to rest for approximately five to fifteen minutes in a room temperature environment. Transfer 100 µL of cells from the “-plasmid” tube to the agar plates marked with “LB/Amp -plasmid” and “LB -plasmid.” Take an inoculating loop and bend one of the ends into a 90 degree angle so that the loop now resembles a hockey stick. Use the bent inoculating loop to spread the cells along the plate’s surface while making sure that you are not piercing the agar’s surface. Repeat the same process when transferring cells from the “+plasmid” tube to the plates labeled “LB/Amp +plasmid” and “LB +plasmid.” Once all of the steps above have been completed, stack the plates upside down and wrap them securely with tape. Place the stack of plates in the incubator and incubate for 24-36
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
...q DNA polymerase to each tube while disallowing the tubes to cool and without taking
placed in each tube. Each tube was then placed in a water bath of the
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
Next, label the three test tubes A, B, and C. Spit saliva into the test tubes until there is a relatively sufficient amount, which is about 1-2 mL of saliva for each test tube. After, put two mL of vinegar into test tube A. Put two mL of distilled water in both test tubes B and C. Thump the tubes, or repeatedly pushing on it with the index finger, to let the solution mix together. Then, treat test tube B into the boiling water bath for five minutes. After the five minutes are over, remove the test tube from the bath and put it back onto the test tube rack. Next, put five mL of the starch solution to all three tubes and thump all the tubes to combine.
5.) One at a time, place your test tubes in the water bath and heat the first test tube to 25 , the second to 50 , the third to 75, and the last to 100 degrees c. Remeber to stir with your stirring rod every so often.
From the direct count culture, a serial dilution was completed, and this is where the final dilution had the cell quantity in the countable range of 30-300. For viable count, two Petri dishes were labeled, with “A (1 x 10 ^ -5)” and one “B (2 x 10 ^ -5),” To add, the first 99 mL DI H2O bottle was labeled with “1 to 100” ratio and the other bottle was labeled with a “1 to 10,000” ratio. Lastly, the 9 mL DI water tube was labeled with “1 to 100,000” ratio. Researchers added 1 mL of pure yeast culture into a bottle labeled “1:100” and mixed well. After, they took 1 mL from bottle number 1 and put it into bottle number 2, also mixing well with the 99 mL DI H2O. Taking 1 mL of bottle number 2, researchers added this amount to the 9 mL water tube and mixed well.
Spread plates are mainly used with a diluted solution or mixed with microbes so that individual colonies can be isolated. Spreading 0.5ml of a sample over the agar jelly with a L shaped glass spreader to ensure that the bacteria is spread as even as possible over the full surface of the agar plate to separate the bacteria. This method is used to count the number of colonies on the surface one advantage is that cultures are never exposed to more than 45oC . Contamination Cross contamination in the health care setting can happen in a number of different ways, from eating and drinking, smoking, applying cosmetics, preparing food, performing clean r sterile procedures, working with patients.
As the test tubes were taken out of the water bath and placed the in
Answer: Chromosomal DNA became renatured and entangled with cellular debris after addition of precipitation buffer (N4), whereas, centrifugation step separated the chromosomal DNA from the plasmid containing supernatant.
2. In the large beaker, put water and boil it completely. After that, remove the beaker from heat. 3. Sample tubes (A-D) should be labeled and capped tightly.
- After each trial, leave equipment for 2 minutes to cool. After this when moving hot test tubes and beakers, use the heat proof gloves provided to prevent burning skin
By using an inoculating loop, one loop of S41 is inoculated into 2 conical flasks of YEPA broth, YEPD broth and YEPPO broth respectively.
We decided to use 2 minutes, as this gave the best results. We repeated the experiment fifteen times, firstly heating 50cm3 three. times, then add 25cm3 of water each time, so from 50cm3 to 350cm3. After each repetition we rinsed the beaker so as to make this a fair. experiment.