Antibiotic Concentrations for Molecular Biology

Cloning Antibiotic Concentrations

 

Dissolved and Stored in Water: Add 1µl per 1ml of media

 

Kanamycin – Stock 50mg/ml, final concentration 50µg/ml
Ampicillin - Stock 100mg/ml, final concentration 100µg/ml (50µg/ml can also be used, but more satellite colonies will appear)
Streptomycin – Stock 50mg/ml, final concentration 50µg/ml  
Spectinomycin - Stock 50mg/ml, final concentration 50µg/ml

 

Dissolved and Stored in Ethanol: Add 1µl per ml media
Chloramphenicol - Stock 25mg/ml, final concentration 25µg/ml

 

Dissolved and Stored in Methanol: Add 1µl per ml media
Tetracycline HCL - Stock 10mg/ml, final concentration 10µg/ml

 

Author: Dr Ryan Cawood 

 

 

Bacterial Media Recipes

 

Quick links...

2xYT BrothLB Agar,  LB BrothLB (Low Salt) BrothSOB MediaSOC Media,  Terrific Broth

 

When making media it is often useful to make it in a larger bottle than you plan to autoclave it in because some of the media may come out of the bottle on autoclaving. Always loosen the lid of a bottle when autoclaving the media, otherwise the bottle may break/explode.

 

2xYT Media (1 Litre)

  • Measure out 750ml of distilled H2O.

Add:

  • 16g Bacto Tryptone.
  • 10g Bacto Yeast Extract.
  • 5g NaCl.
  • pH to 7.0 with 5M NaOH.
  • Make up to 1 litre with distilled H2O.
  • Sterilize media by autoclaving.

 

 

LB Agar (1 Litre)

  • Measure out 750ml distilled H2O

Add:

  • 10g Bacto-tryptone
  • 5g Yeast extract
  • 10g NaCl
  • pH to 7.5 with 5M NaOH
  • Add 15g agar
  • Adjust the volume to 1 litre with distilled H2O
  • Sterilize the media by autoclaving



 

LB Broth (1 Litre)

  • Measure out 750ml distilled H2O

Add:

  • 10g Bacto-tryptone
  • 5g Yeast extract
  • 10g NaCl
  • pH to 7.5 with 5M NaOH.
  • Adjust the volume to 1 litre with distilled H2O
  • Sterilize the media by autoclaving

 

 

LB Broth (Low Salt) (1 Litre)

  • Measure out 800ml of distilled H2O

Add:

  • 10g Bacto-tryptone
  • 5g Yeast extract
  • 5g NaCl
  • pH to 7.0 with 5M NaOH
  • Adjust the volume to 1 litre with distilled H2O
  • Sterilize by autoclaving

 

 

SOB Media (1 Litre)

  • Measure out 750ml of distilled H2O

Add:

  • 20g Bacto Tryptone
  • 5g Bacto Yeast Extract
  • 2ml of 5M NaCl
  • 2.5ml of 1M KCl
  • 10ml of 1M MgCl2
  • 10ml of 1M MgSO4
  • pH to 7.0 with 5M NaOH
  • Adjust the volume to 1 litre with distilled H2O
  • Sterilize by autoclaving

 

 

SOC Media (1 Litre)

  • Measure out 750ml of distilled H2O

Add:

  • 20g Bacto Tryptone
  • 5g Bacto Yeast Extract
  • 2ml of 5M NaCl
  • 2.5ml of 1M KCl
  • 10ml of 1M MgCl2
  • 10ml of 1M MgSO4
  • 20ml of 1M glucose
  • pH to 7.0 with 5M NaOH
  • Adjust the volume to 1 litre with distilled H2O
  • Sterilize by autoclaving

 

 

Terrific Broth (1 Litre)

Terrific broth is made in two separate solutions that are combined after sterilisation. 

 

Media base solution:

  • Measure out 750ml of distilled H2O

Add:

  • 12g Tryptone
  • 24g Yeast extract
  • 4ml Glycerol
  • Make the volume up to 900ml with distilled H2O
  • Sterilize by autoclaving
  • Allow the media to cool to room temperature
  • Measure 90ml of distilled H2O.

 

Phosphate Buffer Solution:

  • Measure 90ml of distilled H2O
  • Add 2.31 g KH2PO4 monobasic
  • Add 12.54 g K2HPO4 dibasic (for trihydrate 16.45 g)
  • Adjust the volume to 100 mL with distilled H2O
  • Sterilise by either filtration or autoclaving

 

Add the Phosphate buffer to the media base solution but only after the media has cooled to at least 60ºC.

 

Ligase Buffer Recipe

10x Ligation buffer

0.5M Tris-HCl (pH 7.6)
100mM MgCl2
100mM dithioltheritol (DTT)
500µg/ml bovine serum albumin (BSA)
Made up in water

Tip: This buffer is stored in small aliquots at -20°C.

Note: The BSA can precipitate after freezing and should be re-suspended by vortexing or warming to 37°C before use.

 

 

TAE and TBE Electrophoresis Buffers

Tris Borate EDTA

Recipe makes 10x concentrated TBE buffer (1 litre):

 

108g Tris base
55g Boric acid
40ml 0.5M EDTA (pH8.0)
Make up to 1 litre using deionised water

 

 

Tris Acetic Acid EDTA

Recipe makes 50x concentrated TAE buffer (1 litre):

 

242g Tris base
57.1ml Glacial Acetic Acid
100ml 0.5M EDTA (pH8.0)
Make up to 1 litre with deionised water

 

Tip 1: For larger DNA fragments (>10 kb) TAE is recommended as the image resolution may be slightly better. TAE has a lower buffering capacity compared to TBE.

 

Tip 2: For small DNA fragments (<1 kb) TBE is recommended as the image resolution is better. It has a higher buffering capacity than TAE.

 

Tip 3: Avoid using buffers more than 3 times. As time goes by the water evaporates and increases the concentration of the buffer components. For this reason don’t use too many new gels in old buffers as the concentrations will be different between the two and cause an imbalance in the conductivity.  

 

Note 1: TAE heats up more than TBE which can cause damage to gels and equipment on regular long (>4 hr) runs.

 

Note 2: Some gel extraction kits recommend one buffer and may work more efficiently with that buffer. Check gel extraction kit manuals carefully. Most kits seem to have no buffer preference.

 

 

Agarose Gel Loading Dye

Recipe makes 4x Loading Dye: (For general agarose gel use)
24% sucrose
0.1% bromophenol blue
40mM EDTA

 

Note: Bromophenol blue can get everywhere. Be very careful with the powder as even small amounts can cause problems. Your lab collegues will not be happy if they find everything turns blue when they add water to it.

 

Author: Dr Ryan Cawood

 

 

DNA Storage Buffers

TE buffer: (for standard DNA manipulations)

10mM Tris-HCl pH 8.0
0.1mM EDTA

 

Tip 1: DNA is quite stable in TE buffer at 4ºC. If stored in elution buffer or water then freezing at -20 ºC is advised.

 

Note 1: TE can reduce some enzymatic reactions, for example, DNA sequencing reactions and occasionally ligations. Elute DNA to be ligated or sequenced in either elution buffer or water.   

 

 

Elution Buffer (Used by many of the kits from major suppliers)

10mM Tris-Cl, pH 8.5

 

Note: Elution buffer is basically TE without the EDTA which is the component that can disrupt enzymatic reactions. DNA is more stable in this than in water.

 

 

Nuclease Free water

To make nuclease free water, fill a small clean lab bottle with deionised water filtered through a 0.22µm pore filter using a syringe and then autoclave. Replace every few weeks/months to avoid contamination.

 

Note 1: Tissue culture grade nuclease free water is available from many suppliers. It is not that expensive (<£2) but you can just make it yourself. You will only be using small amounts so if you do buy it will last you a while if it doesn’t become contaminated.

 

 

Making LB Broth

This recipe is designed to make 1 litre of Luria-Bertani (LB) media for growing E. Coli with standard plasmids. To watch the video of this protocol click here.

 

10 g Bacto-Tryptone
5 g Bacto-Yeast extract
10 g NaCl  
Up to 1 litre with water

 

Potential Hazard: Powders can be sensitising and you can develop allergies to them. Avoid inhalation of aerosolised powder.

 

 

Protocol

Measure out the amounts above and add to a 1 litre bottle with a good working lid (see note below). Fill with clean distilled water (ideally at least 18 megaohms). Tighten lid and shake to mix the liquid and powder, don’t expect to dissolve it all but simply to free the powder from the bottom and remove the major clumps. If not mixed properly the powder can bake on the bottom of the bottle. Undo the lid about half a turn; add some autoclave tape and then autoclave.


After autoclaving make sure the lid is tightened back up. Ideally the lid is tightened whilst the bottle is warm/hot as this will create a vacuum as the liquid cools. When cool add the correct amount of antibiotic (see below) and store at 4ºC. It can be left in the cold room for a few months but watch out for fungus contamination and a decline in antibiotic activity over time. To maintain sterility it is best to add the antibiotic and take aliquots out of the bottle in a class 1 or 2 laminar flow hood.

 

 

Notes and Tips:

Tip 1: Many lab bottles have a blue or clear plastic rim which can go missing or get burnt by flaming. These blue rims prevent the liquids running down the bottle when pouring and keep an air tight seal after autoclaving. Make sure the rim is intact.

 

Tip 2: 1 litre is quite a lot of media and antibiotic, scale down the recipe to suit your needs. I generally make 0.5 litres at a time.

 

Note 1: Antibiotic concentrations


Antibiotic concentrations

Dissolved and Stored in Water: add 1µl per 1ml of media
Kanamycin – Stock 50mg/ml, final concentration 50µg/ml
Ampicillin - Stock 100mg/ml, final concentration 100µg/ml (50µg/ml can also be used, but more satellite colonies will appear)
Streptomycin – Stock 50mg/ml, final concentration 50µg/ml  
Spectinomycin - Stock 50mg/ml, final concentration 50µg/ml

 

Dissolved and Stored in Ethanol: Add 1µl per ml media
Chloramphenicol - Stock 25mg/ml, final concentration 25µg/ml

 

Dissolved and Stored in Methanol: Add 1µl per ml media
Tetracycline HCL - Stock 10mg/ml, final concentration 10µg/ml

 

Note 2: Pre-mixed LB powders are available from many suppliers and don’t actually work out much more, if any, expensive.  

 

Note 3: For larger plasmids (>50Kb), growing competent cells or cosmids, a richer broth such as 2xYT may be more useful.

 

Author: Dr Ryan Cawood