How Much Ampicillin To Add To Lb Broth

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Introduction

Plasmids tin can deport 1 or more than antibiotic resistance genes, which confer resistance to a specific antibiotic to the leaner carrying them. The presence of an antibody resistance gene on a plasmid allows researchers to easily isolate bacteria containing that plasmid from bacteria that do not comprise information technology by artificial selection (i.e. growing the bacteria in the presence of the antibiotic).

Luria broth (LB) is a food-rich media commonly used to civilization bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather diet from the LB within. The addition of an antibiotic to this gel allows for the choice of just those bacteria with resistance to that antibiotic - unremarkably conferred by a plasmid carrying the antibiotic resistance factor. The post-obit protocol will permit y'all to brand your ain LB/agar plates with your antibody of interest.

Last Upload: September 14, 2016

Protocol Video

Before you begin this protocol, lookout the protocol video below to get a quick idea of how the whole process works.

Equipment

• Autoclave
• H2o bath
• Pipetman

Reagents

• 37 g pre-mixed pulverization consisting of:
  • 5.0 k yeast excerpt
  • x.0 g peptone from casein
  • x.0 g sodium chloride
  • 12.0 m agar-agar
• 1 L Sterile H_iiO
• Sterile plates of your desired size - we commonly utilise threescore mm x xv mm plates which tin can hold 5-x mL of agar and on which you can individually distinguish a maximum of ~100 colonies

  

  *Pro-Tip* If you will be screening a large number of colonies, we recommend using larger plates. Many labs use 100 mm x 15 mm plates which tin can hold 10 - 15 mL of agar.

• Autoclavable flasks
• Sterile pipettes
• Water ice bucket to agree antibiotic
• Antibiotic at 1000 x concentration disolved in the advisable liquid solvent. Encounter tabular array to the right for appropriate antibiotic concentrations.

Antibiotic Concentrations

Antibiotic	Recommended Stock Concentration	Recommended Working Concentration
Ampicillin	100 mg/mL	100 µg/mL
Bleocin	five mg/mL	5 µg/mL
Carbenicillin*	100 mg/mL	100 µg/mL
Chloramphenicol	25 mg/mL (deliquesce in EtOH)	25 µg/mL
Coumermycin	25 mg/mL (deliquesce in DMSO)	25 µg/mL
Gentamycin	10 mg/mL	10 µg/mL
Kanamycin	50 mg/mL	50 µg/mL
Spectinomycin	50 mg/mL	50 µg/mL
Tetracycline	ten mg/mL	10 µg/mL

Note: Unless otherwise indicated, the antibody pulverisation can be dissolved in dH_iiO.

*Note: Carbenicillin tin can be used in place of ampicillin. Carbenicillin is more stable, so information technology is potentially more effective at selecting only leaner containing the plasmids of interest (for instance, fewer satellite colonies volition grow). However, it is also more than expensive.

Procedure

1. Mensurate 37g of pre-mixed LB-agar powder per L of molten agar you'd similar to make. The precise mass you measure out will be based on the number of plates you'd similar to cascade.

   For case: Because we'd like to make 20 plates, and our plates tin can hold a maximum of ten mL, we'll desire 200 mL of media total.

   Importantly, we'll actually brand a fleck more than 200 mL (~220 mL) just in case nosotros spill annihilation or have small errors in measurement. You should also ever make a flake more gel-ager mix than y'all retrieve you'll need.

   Then for 220 mL of LB-agar, we'll need:

   (37 g pre-mixed LB-agar powder/L) x (0.220 L) = 8.14 g pre-mixed LB-agar powder.

2. Transfer the LB-agar pulverization you've measured out into an accordingly sized bottle for autoclaving. We make 400 mL of agar in 1 Fifty 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.
3. Transfer the sterile water (in our case 220 mL) to the aforementioned bottle and swirl to grade a medium/agar colloid.
4. Cover the opening of the bottle with its cap or aluminum foil (but practise not brand an air-tight seal!) and record the bottle with autoclave tape. The autoclave record will darken during the autoclave procedure if your sample has spent at to the lowest degree 10 min at 121 ℃. Utilize lab record to label the bottle with your initials, the date, and the canteen contents. This will clear upwardly whatever confusion later if your forget your bottle in the autoclave.
5. Place the gel mix in the autoclave and run on a setting that gets the sample to at least 121 ℃ under xx psi for at least thirty min. The high pressure will foreclose your gel mix from boiling over at loftier temperature.

   

   *Pro-Tip* Although our recommended temperature should kill most potential contaminants, certain types of spores will stubbornly survive even nether the harsh weather condition in the autoclave. Exist sure to cheque the literature for an advisable sterilization technique if you are working with any weird and wonderful organisms.

6. While your samples are sterilizing in the autoclave, you should prepare your plate pouring station:
   • Discover an empty department of lab bench with a working flame.
   • Spray down the bench with a 70% ethanol solution and wipe downwards with a paper towel.
   • Count out the appropriate number of plates and stack them on your lab bench.
   • Label the plates with the date and the medium they will contain including the identity of the antibiotic.

     

     *Pro-Tip* Nosotros batch label our plates with colored marking - detail colors stand for to item antibiotics.

   • Position the flame merely to the side of where y'all'll exist pouring your plates - be sure to go out room for your bottle of molten gel mix, a tube rack containing the appropriate antibiotics, and a section for active pouring.
7. Prepare your antibiotics. Prior to calculation your antibiotic to the molten gel mix, you should create a 1000x stock solution.

For example: If yous'll be preparing plates with a terminal concentration of 100 ug/mL ampicillin, you should make a stock solution of 100,000 ug/mL (100 mg/mL). Simply mensurate 100 mg of ampicillin powder, add information technology to ane mL of water, dissolve by vortexing, and filter sterilize.

8. Gear up a water bath at 60 ℃ with sufficient water to submerge ~75% of the bottle containing your molten gel mixture.

   

   *Pro-Tip* You will absurd the molten agar in the water bath prior to calculation the antibody. threescore ℃ is a good temperature because the molten agar will remain liquid at this temperature just almost antibiotics will not break down at this temperature. Bank check with your antibody's manufacturer to make certain this is truthful in your case.

9. Recall your molten agar mix from the autoclave.

   

   *Pro-Tip* Once your autoclave cycle is complete, we recommend opening the door to the autoclave just a cleft and leaving it open up that style for ~10 min. This will allow any steam to escape from the autoclave and will cool your gel-mix slightly. Yet, you should always use thermally insulated gloves when removing anything from the autoclave.

10. Partially submerge your molten gel-mix in the lx ℃ water bath.



*Pro-Tip* Yous should get out the molten gel-mix in the water bath for at to the lowest degree 5 min. Practice not let any of the water bath water touch the cervix or top of the bottle as this water is not likely to be sterile. Cooled agar should be warm to the touch; as a rule of thumb, if you cannot take the molten agar out of the water bath wearing only lab gloves, it'southward non likely absurd enough to add together antibiotic to. To be sure your agar is at the right temperature, nosotros recommend using a laser thermometer.

11. Light the flame at the plate pouring station and dilute your antibody into your ~60 ℃ molten gel mix using sterile technique.
12. Swirl the agar bottle to ensure even distribution of the antibiotic throughout the agar.
13. Open 1 plate at a time side by side to the flame and begin pouring. Measure your desired corporeality of agar with a pipete for the first plate to get a practiced thought of what that volume looks like in your particular plate.
14. For the balance of the plates, cascade straight from the canteen.

    

    *Pro-Tip* Be certain to swirl your plates afterward pouring to remove bubbles and ensure an even distrubtion of agar over the bottom of the plate. Cap each plate afterward pouring and stack every bit you pour.

    

    *Pro-Tip* If your agar partially solidifies in the bottle while y'all're pouring, you lot should stop pouring and re-make the gel-mix. If you're making plates without whatsoever antibiotic y'all can alternatively re-liquefy the agar by running it through the autoclave again or by microwaving (if you microwave, beware of over-humid!).

15. Leave your plates out on the bench to solidify.

    

    *Pro-Tip* Information technology takes roughly 30 min for our plates to solidify at room temperature, yet we go out them out at room temperature overnight to let them to dry. Afterward overnight drying, we place the plates in a plastic bag with an absorbent material to reduce condensation. The plates are then stored at four ℃ until apply.

16. Once your plates have solidified and dried, you should test them to brand sure the antibody functions properly:
    • Take out two plates.
    • On the offset plate, streak out a strain that you know to be resistant to the antibiotic.
    • On the 2d plate, streak out a strain that's not resistant to the antibody.
    • Incubate both plates overnight at the appropriate growth temperature and bank check for growth. Meet our sample data section below for positive and negative examination results.

Sample Data

In all cases below (-) indicates that the tested strain is not supposed to be resistant to the antibiotic, (+) indicates that the tested strain is supposed to be resistant to the antibiotic.

Positive Consequence: Only the Resistant Strain Grows

If everything was prepared properly, you should see growth of only the resistant strain. If you get this last result, your plates are ready for use or storage at 4 ℃. You should non store your plates for longer than 1 month at any temperature and should always cheque for contamination prior to use.

Negative Result 1: Both Strains Grow

Assuming the appropriate strains were streaked on the appropriate plates, so if both strains abound, it'southward possible that:

1. The antibody broke down.
2. You forgot to add the antibiotic.
3. You added the antibiotic at likewise low a concentration for selection.

Negative Result 2: Neither Strains Grows

If neither strain grows, information technology'due south possible that:

1. You used the wrong antibiotic.
2. Your antibiotic is at as well high a concentration.
3. One or both of your strains are no longer viable.
   • You tin can check for this possibility by streaking out both strains on plates without any antibiotic.

Negative Result 3: Simply the Non-resistant Strain Grows

If only the non-resistant strain grows, it'due south likely that you've used the incorrect antibiotic, or confused your strains.

Source: https://www.addgene.org/protocols/pouring-lb-agar-plates/

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