How do you prepare X-gal?

How do you prepare 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 dilute X-gal?

(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.

Can you dissolve X-gal DMSO?

X-Gal is soluble in organic solvents such as DMSO and dimethyl formamide, which should be purged with an inert gas. The solubility of X-gal in these solvents is approximately 30 mg/ml. X-Gal is sparingly soluble in aqueous buffers.

How do you make a BLUO gal stock solution?

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. It is not necessary to filter sterilize these solutions.

Is XGAL toxic?

Acute toxicity : Oral: Harmful if swallowed. Dermal: Harmful in contact with skin. Inhalation: Harmful if inhaled. Potential adverse human health effects and symptoms : Dust in eyes may cause mechanical irritation.

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.

What is blue white colony screening?

Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose.

How do you make agar amps with LB plates?

Protocol

1. 500 mL makes about one sleeve of petri dishes.
2. Add 25g LB Broth per Liter to an appropriately sized beaker.
3. Add Water and Stir until clumps are gone.
4. Add 15 g Agar per Liter , this will not dissolve.
5. Transfer to a erlenmeyer flask at least 2x the volume that you are making.

Is IPTG an inducer?

IPTG (isopropyl beta-D-thiogalactoside) Inducer for Beta-Galactosidase Expression acts as a molecular mimic of a lactose metabolite. The presence of IPTG triggers the activation of the lac operon for downstream gene transcription due to its binding the lac repressor.

How long do LB Ampicillin plates last?

This recipe is for 500 mL of LB agar. This makes about 20 plates (1 bag). IMPORTANT NOTE: Ampicillin and carbenicillin stocks must be kept in the -80 C. They only last for 3-6 months if they are made from the sodium salt.

How do you know if a bacteria has taken up a plasmid?

Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony. Several colonies are checked to identify one with the right plasmid (e.g., by PCR or restriction digest). A colony containing the right plasmid is grown in bulk and used for plasmid or protein production.

What is insertional activation?

Definition. Insertional activation/inactivation refers to either activation of an endogenous gene which is located near an integrated transgene, or to disruption of a gene or other functional sequence by insertion of a transposable element.