Personally I would not recommend using plates more than four months either. Or its the first hypothesis where you are not keeping selective pressure for the plasmid with antibiotics. Background colonies are also somewhat dependent on the host strain you are using. I get tons of plasmid from the rest of the culture. Use the device to touch a colony of bacteria from a petri dish or test tube. The students should be instructed when opening the packets to touch only that part of the object that will not come in contact with the solutions or petri dishes. The growth of colonies that do not contain a resistance marker suggest a problem with your selective conditions.
I found out today that I missed out on 3 ingredients. If you pick a couple of these colonies you will probably find that they do not grow in your liquid media with Kanamycin present as it is easy to add antibiotic to cold liquid so no degredation of kanamycin occurs. Cover with foil and allow to sit overnight and store in cold room. Proceed to the Recombinant Transformation Day 4 of the lab when your teacher instructs you to do so. Store the plates at 4°C A quick way to label your plates is to have a color code for each antibiotic and medium type you tend to use e.
Autoclave for 25 minutes After autoclaving you can of course store the medium-agar mix in a toughened glass bottle then melt it in a microwave or water bath when needed. Mark the plates with a single purple line on the side. Now remove the lid and put it between the two plates, so it sits on top of both, covering each by about half. Place the required volume of distilled water in one or more glass bottles with caps. Use a fresh pipette tip for each reagent.
Aliquot into 50 ml tubes and freeze at -2 degrees C 4. The original method calls for growing the overnight E. Add the antibiotic and pour immediately into the plates. In Day 3 of the student lab, the four fragments from the digests are ligated together to form recombinant molecules. Then place the bottle in a boiling water bath and sterilize according to Item 3a.
Unused broth can be reboiled and stored in the refrigerator for future use. Use MathJax to format equations. With those assumptions in mind: 1. It is best to prepare a few extra plates for the entire class in case contamination occurs in one or more of them. Also, when you say you have changed the Kanamycin stock, does that mean you went back to the freezer and grabbed a new but previously made-up aliquot? The antibiotic degrades over time, making it ineffective at selecting transformed bacteria grown on the plate.
The autoclave is fine cuz others in the lab are using the same autoclave and not getting contamination on their plates. Place on ice for 20 minutes. Timing the events in the lab is very important. If there are any bubbles in the plates, briefly pass the flame over to pop them. Place cells on ice in the refrigerator approx. Buffer A 10X For 100 ml: 7. These colonies are much smaller than my dh5a colonies and do not carry any plasmid.
With the caps loose, sterilize the solution by one of the methods described for the calcium chloride Step 3a, b, or c. Saves time and effort of having to clean the insides of the microwave. Add 100 ml of sterile solutions of 0. Thanks for contributing an answer to Biology Stack Exchange! Priyamvada Jain - Kan is not prone to satellites as Amp is - by far - but it can still happen. You can ethanol-precipitate plasmids and then redissolve it in water.
After the salts have been dissolved, adjust the volume of the solution to 100 ml with deionized water. However, many people are quite conservative and would not use antibiotics plates more than a month old. Mid-Log Suspension: Ideally, the mid-log suspension should be prepared 3-4 hours before class on Day 2. The alternative is to continue to incubate the mixture overnight. You should plan six uninterrupted class periods of 45-50 minutes each to complete the exercise.
Human insulin production, so that you don't have to harvest humans, and extracting it from pigs or cows is very expensive and resource intensive. Before I used a waterbath, I used to just cool it in air but would inevitably forget about it and come back to find solidification had already started — lumpy plates are no good for spreading! Posts: 44 Joined: June 29th, 2012, 1:56 pm It would be useful to know what the experiment was. This allows space for rising bubbles while melting the agar in a microwave. I then started liquid cultures of a few of those colonies and extracted the plasmid, all is good so far. Alternatively - how did you save the liquid culture? Make the 5% solution by adding 1 ml of sterile, cool distilled water to the 0.
If that description fits your case, your small spurious colonies are most probably satellites. Sterilization of packets of toothpicks, glass pipettes if appropriate , and paper clips can be accomplished by wrapping each item in aluminum foil, labeling the contents with a marking pen, and a baking them in an oven at 350°F for 15 minutes, or b putting them in a pressure cooker at 15 pounds for 15 minutes, or c placing them in an autoclave for 15 minutes. Do not touch the part of the paper clip that comes in contact with the agar. If the agar solidifies, it cannot be reheated because the ampicillin will be destroyed above 60° C. Ampicillin loses about 10% activity at 4 weeks.