How do you prepare LB ampicillin IPTG X-gal plates?

How do you prepare LB ampicillin IPTG X-gal plates?

(or 2 µl X-Gal Solution (100 mg/ml) per 1 mL of Media). Add 10 µl IPTG (100mM) per 1 mL of Media for a final concentration of 1mM. Add screening antibiotic of choice (Ampicillin, Kanamycin, Carbenicillin, etc). Pour plates and allow to cool to room temperature (usually at least 30 minutes) before use.

What is the purpose of the IPTG and X-gal on the LB plates?

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. IPTG is a non-metabolizable analog of galactose that induces the expression of lacZ gene. It should be noted that IPTG is not a substrate for β-galactosidase but only an inducer.

How do you make LB agar plates?

Dissolve 15 g of Bacto agar in 1.0 L of LB medium and sterilize by autoclaving. Cool to 50°C in a temperature-controlled water bath. Add 0.50 mL of 100 mg/mL ampicillin. Pour into plates.

How do you dissolve X-gal?

Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. Use a glass or polypropylene tube. Wrap the tube containing the solution in aluminum foil to prevent damage by light and store at -20°C. It is not necessary to sterilize X-gal solutions by filtration.

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 do you identify contamination in LB agar plates?

If the plate has not been inoculated, the presence of any bacterial colonies indicates contamination. On an inoculated plate, look for colonies that display morphology different than what you would expect from the type of bacteria used to inoculate the plate.

How do you prepare pound media?

How to make a 1 L LB media solution

1. Weigh out 10 g tryptone, 10 g sodium chloride (NaCl) and 5 g yeast extract and add to a 1 L Duran bottle.
2. Measure out approximately 900 mL of distilled water and add to the Duran bottle.
3. Shake the bottle to dissolve the reagents.

How do you make LB agar plates from LB broth?

Preparation of LB agar plates: (~30-35 plates) In a 1L autoclave bottle (orange cap), add: 37g LB Agar powder 1000mL MiliQ water • Swirl to mix. Powder may not dissolve completely, that is ok but avoid clumps. Add a fresh piece of autoclave tape.

How long does XGAL last?

6-12 months
X-Gal Solution is stable for 6-12 months at -20°C. However, frequent use will cause degradation of the solution over time and we recommend making new stocks every 2-3 months depending on use.

How do you dissolve a BLUO gal?

IPTG can be reconstituted in water. Make a stock of 100 mM in water and store working aliquots at -20°C. X-gal can be reconstituted in DMSO, or in a 50:50 mix of DMSO and water. To do the latter, you must dissolve in DMSO first, and then add water to bring up to final volume.

How do you make LB agar ampicillin plates?

What is the difference between LB agar and LB broth?

Agar is a polysaccharide isolated from a plant and is used for microbial cultures on solid media. LB broth is generally used for inoculum preparation or start cultures in suspension medium. The only difference between broth and agar media is that broths do not contain an agar component… Thank you.

How to prepare X-gal LB agar plates for blue/white colony screening?

This protocol is for the Preparation of X-Gal/IPTG LB Agar Plates for Blue/White Colony Screening. For individual LB (Luria Broth) agar plates: Pour sterile warm LB agar (about 25 mL) into a Petri dish. Dry opened LB plates at room temperature under UV light for about 30 minutes.

How do you prepare Luria broth agar plates?

For individual LB (Luria Broth) agar plates: 1. Pour sterile warm LB agar (about 25 mL) into a Petri dish. 2. Dry opened LB plates at room temperature under UV light for about 30 minutes. 3. Add 40 μL of the Thermo Scientific X-Gal Solution (20 mg/mL), ready-to-use (Cat #R0941).

How much X-gal do I add to LB agar?

For batch use, add the following directly per 1 mL of the liquid LB agar (kept at about 50 °C): 6. 1 μL of X-Gal Solution (20 mg/mL), ready-to-use.

How do you measure the mass of a plate of agar?

Measure 37g of pre-mixed LB-agar powder per L of molten agar you’d like to make. The precise mass you measure out will be based on the number of plates you’d like to pour. For example: Because we’d like to make 20 plates, and our plates can hold a maximum of 10 mL, we’ll want 200 mL of media total.