How much kanamycin do you put on a LB plate?
Autoclave 1 L of LB agar. Cool to 55°C. Add 1 mL of the kanamycin stock (i.e., 50 mg).
What is LB kanamycin?
Miller′s LB is a highly-referenced microbial growth medium used for the cultivation of E. coli. This nutrient-rich microbial broth contains peptides, amino acids, water-soluble vitamins, and carbohydrates in a low-salt formulation. The addition of agar provides a solid medium for microbial growth.
How do you make LB agar with kanamycin?
Kanamycin – add 1 ml kanamycin stock (50mg/ml) per liter of agar so the final concentration is 50µg/ml. Mark the plate with “K” on the side. 3. Chloramphenicol – add 1 ml chloramphenicol stock (25mg/ml) per liter of agar so the final concentration is 25µg/ml.
How do you make LB agar plates with ampicillin?
To make 500 ml of LB agar containing 50 µg/ml ampicillin, add 2.5 ml of 10 mg/ml ampicillin stock. Ensure LB agar is cooled to 50°C before adding ampicillin, excessive heat will degrade the ampicillin. Swirl or use the stir bar and a stir plate to mix the ampicillin into the agar taking care not to introduce bubbles.
How long do kanamycin plates last?
The sealed plate can be stored at 2-8 °C for about one month. If the plate is not sealed to prevent evaporation of moisture, the kanamycin can degrade.
How much antibiotics add to agar plates?
Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium. The concentration of antibiotic required for effective over-agar selection has been empirically determined.
What does kanamycin do to bacteria?
Kanamycin is an aminoglycoside antibiotic. Aminoglycosides work by binding to the bacterial 30S ribosomal subunit, causing misreading of t-RNA, leaving the bacterium unable to synthesize proteins vital to its growth.
What does LB agar stand for?
According to Bertani, LB has been variously misconstrued to stand for “Luria Broth”, “Luria-Bertani” medium, and “Lennox Broth”; however, the acronym originally stood for “Lysogeny Broth” (Bertani, 2004). The agar form of the medium should be designated LA but it is often referred to as LB.
How do you make 500ml LB agar?
Protocol
- 500 mL makes about one sleeve of petri dishes.
- Add 25g LB Broth per Liter to an appropriately sized beaker.
- Add Water and Stir until clumps are gone.
- Add 15 g Agar per Liter , this will not dissolve.
- Transfer to a erlenmeyer flask at least 2x the volume that you are making.
How much ampicillin do I add to agar plates?
Ampicillin – add 1ml ampicillin (at 100mg/ml) per liter of agar to obtain a final concentration of 100ug/ml. Mark the plate with a single red line on the side.
Does kanamycin expire?
Kanamycin sulfate is a sterile filtered solution formulated to contain 10mg/ml Kanamycin. Storage and Shelf Life: Store at 2 – 8°C. Shelf life of the product is 24 months.
How to make molten agar with LB-agar powder?
Procedure Measure 37g of pre-mixed LB-agar powder per L of molten agar you’d like to make. Transfer the LB-agar powder you’ve measured out into an appropriately sized bottle for autoclaving. Transfer the sterile water (in our case 220 mL) to the same bottle and swirl to form a medium/agar colloid.
How big of a bottle do you need for LB agar?
Transfer the LB-agar powder you’ve measured out into an appropriately sized bottle for autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The extra empty volume is necessary to prevent your molten agar from boiling over in the autoclave.
How do you check for growth on agar plates?
The plates are then stored at 4 ℃ until use. Take out two plates. On the first plate, streak out a strain that you know to be resistant to the antibiotic. On the second plate, streak out a strain that’s not resistant to the antibiotic. Incubate both plates overnight at the appropriate growth temperature and check for growth.
Can you re-liquefy agar plates without an antibiotic?
If you’re making plates without any antibiotic you can alternatively re-liquefy the agar by running it through the autoclave again or by microwaving (if you microwave, beware of over-boiling!). Leave your plates out on the bench to solidify.
