Do you need to autoclave LB agar?
If you preserve your LB in a tightly closed bottle, there is no need to autoclave again. Just microwave and liquify it. In your case, where you stored you LB in a flask- I suppose you covered it with foil or air-permeable fabric, it should be fine if you didn’t open it after autoclave.
What is the purpose of the LB amp agar plate?
What is the purpose of the LB/amp/ara agar plate? It is a selective medium with an antibiotic which prevents growth of non-resistant cells and a sugar that allows for the expression of a plasmid gene.
What grows on LB AMP plates?
LB: This is the standard agar that all bacteria grow on.
How long do LB AMP plates last?
The plates last longer and reduces the formation fo satellite colonies. They last about 3-4 months.
How do you dissolve LB agar?
Microwave the bottle for 10-second stints, until the liquid starts to boil. 3. With autoclave gloves, swirl the bottle to dissolve any remaining agar. Any particulates should be completely dissolved.
How do you autoclave LB agar?
Preparation of LB agar plates: (~30-35 plates) Add a fresh piece of autoclave tape. Autoclave (121°C, 20 minutes) on LIQUID cycle – be sure you add water to the autoclave basin before starting the cycle! After cycle is complete, remove to benchtop to cool to ~50°C.
Why did no bacteria grow on the LB amp plate?
The plasmid does not fluoresce green. The bacterium cannot grow in the presence of the antibiotic ampicillin unless it contains the plasmid, and so there will be no growth on the LB/Amp plate of the bacteria without the plasmid.
How do you grow LB media for bacterial growth?
How to make a 1 L LB media solution
- Weigh out 10 g tryptone, 10 g sodium chloride (NaCl) and 5 g yeast extract and add to a 1 L Duran bottle.
- Measure out approximately 900 mL of distilled water and add to the Duran bottle.
- Shake the bottle to dissolve the reagents.
Why are my colonies so small?
You are only plating too many cells, and due to competition for nutrients, etc. the colonies are small. Try plating 1/10 of what you are using.
How long is LB media good for?
LB broth containing antibiotics should be stored at 4°C and is good for one month.
How do you store LB plates?
Store plates upside down in a refrigerator or cold room. If they are stored in a room, check the plates after a few hours for condensation in the lid. If you have the plates upside down and there is condensation in the lid, there must be some heat source above that is driving water out of the agar and into the lid.
How do you pour agar plates without condensation?
Tips for Pouring and Storing Agar Plates
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- • It is best to pour agar that is about 50º C.
- • If you pour your own agar plates and get lots of bubbles, try gently passing the flame of a Bunsen.
- • To prevent condensation of water in plates, let them solidify and dry at room temperature until the.
- •
What temperature do I pour agar?
Water temperature should remain at around 100°C. Leave it in the water bath until the agar is completely melted. While wearing heat- protective gloves, carefully remove the hot bottle and let it cool to between 75–55°C before pouring.
Why does the LB amp ARA glow?
+pGLO, LB/AMP/ARA: They were exposed to AMP and arabinose sugar, causing bacteria to grow with ampicillin resistance. Colonies glowed due to exposure to arabinose sugar and UV light.
Why do you incubate on ice for 2 minutes?
then the heat shock is to improve the uptake of the DNA into the cells as it changes the fluidity of the membrane and increase pore sizes. last (but I’m not sure why) is the 2-3 mins incubation on ice is to increase the chance as well of the plasmid DNA to enter the cell!!
How long is LB good for?
media like LB is always stored at RT. it can be used for 6 months or even moe, or till you see some turbidity or lesions in the media. That is a normal practice. I also prepare several hundred mL of LB, and store them at room temperature.
Why are some colonies white?
Those colonies containing plasmids with an insert can be differentiated from those without an insert by the color of the colony (white versus blue). The insert disrupted the β-galactosidase gene, and therefore these colonies remain white.
What happens if you incubate bacteria too long?
If a bacterial culture is left in the same media for too long, the cells use up the available nutrients, excrete toxic metabolites, and eventually the entire population will die.
Can I drink LB media?
It should not be ingested. It may cause some irritation to the gut flora, vomitting or indigestion. It is highly unlikely that anyone would waste money on studies to determine the safety of ingestion for LB or any microbiological medium. That is silly.
Can you autoclave LB media twice?
Though it is not necessary to autoclave the media twice because single time autoclaving at 119 degree Celsius is sufficient to seterlize media. If you proceed it further, it will denature the media and may disturb the salt balance, which will effect permieability in microbes and inhibit growth of microbes.