Team:TU Delft/protocols/buffers stock solutions

=Buffers and Stock solutions=


'''Luria-bertani (LB)'''

For 1 liter dissolve in H2O:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|Compound/Ingredient 
|Amount required (1L of medium)
|-
|Tryptone
|10 g
|-
|Yeast extract	
|5 g
|-
|NaCl
|10 g
|-
|Agar+
|15 g
|}

[[*]] Add agar in case a solid medium is required.

Autoclave on a liquid cycle. Once broth reaches room temperature, store in cold room.


'''M9'''

For 1 liter dissolve in H2O:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|Compound/Ingredient 
|Amount required (1L of medium)
|-
|Na2HPO4
|6 g
|-
|KH2PO4
|3 g
|-
|NaCl
|0.5 g
|-
|(NH4)Cl
|1 g
|-
|Agar*
|15 g
|}

[[*]] Add agar in case a solid medium is required.

Once broth reaches room temperature, add to the broth in aseptic conditions the following amount of each solution:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|Solution 
|Amount(1L of medium)
|-
|1 M MgSO4
|2 mL
|-
|1 M CaCl2
|0.1 mL
|-
|20% Glucose (w/v)*
|10 mL
|-
|1M Thiamine-HCl
|1 mL
|}

[[*]] For M9-modified medium no glucose solution is added, instead 10 mL of filtered hydrocarbon/ hydrocarbon mixture are added per liter of medium. Be careful when adding the hydrocarbon/hydrocarbon mixture.

NOTE: Autoclave the solutions 1 M MgSO4, 1M CaCl2, 20% glucose (w/v) (EXCEPT thiamine-HCl 1M) on a liquid cycle. Filter the solution Thiamine-HCl 1M using a 0.22 μm membrane, store the solution in a cold room when is not required.

Store in cold room if the medium is not used.


'''Antibiotics'''

{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|Antibiotic 
|Final concentration in the medium
|-
|Ampicillin
|100 μg/mL
|-
|Kanamycin	
|50 μg/mL
|-
|Chlorampenicol
|35 μg/mL
|-
|Tetracycline
|15 μg/mL
|}

All stock solutions must be kept at -20 ºC and used under aseptic conditions.


'''10x TBE Buffer'''

For 1 liter, dissolve in 800 mL H2O:

{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|Compound/Ingredient 
|Amount required (1L of medium)
|-
|Tris
|54 g
|-
|Boric Acid
|27.5 g
|-
|0.5 M EDTA
|4.65 g or 20 mL
|}

Adjust volume to 1 liter with H2O