Therefore, it is important to pour the plates as rapidly as possible. Pour the next plate and put it next to the first one. Incubate 3 hours in a shaking water bath or 4 hours in the incubator with occasional shaking by hand. Add the appropriate volume of distilled water to the bottle. The protocol has been optimized to fit into a 45- minute classroom session. When the temperature of the tap water reaches 55° C, the contents inside the bottle should be at a similar temperature.
Robin- 3 months is pushing it a bit. Alternatives that yield less than ideal results are: 1 growing bacteria to mid-log 3-4 hours of incubation and then placing them in the refrigerator until the next day, or 2 letting the bacteria incubate on the countertop over night and then placing them in an incubator for a hour or two before use. Proceed to the Recombinant Transformation Day 4 of the lab when your teacher instructs you to do so. Note: These plates are light sensitive. Spread the bacteria on the plate in a zig-zag pattern to obtain individual colonies as the concentration of bacteria on the transfer device becomes less.
Adjust water bath to 37°C. Had kind of the same problem once. Drying the plate is very important for storing the plates and growing colonies on them. Keywords: Antibiotics, Centrifugation, Cloning, Peptide synthesis, Phase transitions, transformation. 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.
Allow the plates to set. I am just sticking my inoculation loop into the liquid culture after sterilizing and cooling and then streaking one quadrant of the petri-dish. The clips should be opened into a 90° angle and the small loop of the clip bent straight. Probably It's Staphylococcus epidermidis or similar. Describe two practical medical or industrial applications of biotechnology other than creating antibiotic-resistant bacteria. Use the plates immediately or seal them. The pAmp digest yields fragments of 3755 base pairs bp and 784 bp, while the pKan digest yields fragments of 2332 bp and 1861 bp.
Ampicillin loses about 10% activity at 4 weeks. You can find useful information about the multitude of E. Finger flick tube to resuspend cells. Real Km transformants can be distinguished from false positives by seeing if they grow flat on the surface of the agar. If midiprep itself is giving a trouble, you could try sidestepping the kits altogether. The following supplies can be shared by groups of three students.
The plates can be kept refrigerated for a month. Make sure that you are not adding the drug to the agar when it is too hot. Place the required volume of distilled water in one or more glass bottles with caps. Also 12-16 hours is a reasonable time for e. Unused broth can be reboiled and stored in the refrigerator for future use. Those plates have been growing for about 10 hours now and I do not see any growth.
Ali Once I became accustomed to the correct volume of agar required in each plate, I now pour my plates in a stack — that is, I pick up my empty stack from the bottom lid, exposing the last base, pour, replace stack, grab next lid up, pour, replace stack… etc. Any details on what you mean by having trouble with getting plasmid? After the packets have cooled, they should be stored unopened at room temperature. The bottom of the dish should be covered with the agar. The product may be shipped up until its expiration date but is generally shipped 2-4 weeks before expiration. The discs are placed right on top of the agar and antibiotic then flows into the surrounding agar resulting in clear zones of inhibition. Adjust the volume of the solution to 1 liter with deionized H2O.
If they colonies are flat, they are not real transformants. With those assumptions in mind: 1. This is not an option if the plasmid needs to be sequenced though. However, many people are quite conservative and would not use antibiotics plates more than a month old. Imidazole For 1 Liter: 102.
Class periods do not have to be consecutive as long as solutions can be stored in the refrigerator or freezer. This allows space for rising bubbles while melting the agar in a microwave. But there are a few finer points that will kill your experiment, make a mess or just cause you inconvenience if you get them wrong. Thanks for contributing an answer to Biology Stack Exchange! One will be used for the fresh bacteria culture used to make mid-log suspension. We have noticed over the past few years that kanamycin is no longer as tight a drug resistance marker as it used to be. Resuspend the pelleted cell by vigorously vortexing the tube with your fingers.
I doubt anything less than 15 ml will cover a plate, though. Have you tried plating out a strain that you are sure is kanamycin sensitive onto some of your plates to see if you observe growth if there are no resistant colonies on the plate? With the caps loose, sterilize the solution by one of the methods described for the calcium chloride Step 3a, b, or c. It works well for many strains commonly used in cloning. . If they were made properly then yes, the double-resistant strains could survive on all the other plates. Place the tube directly back on ice for 1 minute.