LB: Difference between revisions
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== Summary == | == Summary == | ||
L-Broth or LB Medium. | Luria-Bertani Medium (aka L-Broth or LB Medium). (Bertani says LB really stands for lysogeny broth.) LB is a standard growth medium for a variety of bacteria and conditions. | ||
LB contains a milk digest (tryptone) and hence can induce cultures with lac based promoters when they near saturation. | |||
* "LB ... contains enzymatic digests of the milk protein casein (e.g., tryptone or N-Z-amine) and yeast extract. Since milk is rich in lactose, an inducer of the T7 expression system, variable amounts of residual lactose may be present in different lots of these enzymatic digests. These small amounts of lactose do not promote appreciable induction during log-phase growth, but even minute amounts are sufficient to cause induction on approach to saturation, particularly at lower rates of aeration, which allow induction at lower lactose concentration and promote higher levels of induction." -- [http://www.ncbi.nlm.nih.gov/pubmed/24203322 Sturdier 2014] | |||
== Ingredients == | == Ingredients == | ||
#10 g Bacto-tryptone | #10 g Bacto-tryptone | ||
#5 g yeast extract | #5 g yeast extract | ||
#10 g NaCl | #10 g NaCl | ||
# | |||
For plates also add | |||
#15g agar | |||
See also: [[Silver: LB Liquid]] | |||
''Note:'' There are two formulations of LB, Miller and Lennox, that differ in the amount of NaCl. Lennox has less salt, only 5 g/L. The Qiagen miniprep kit recommends LB with 10 g NaCl for highest plasmid yields. | |||
==Protocol== | ==Protocol== | ||
#Mix dry ingredients and add distilled water up to 1 Liter | #Mix dry ingredients and add distilled water up to 1 Liter | ||
| Line 11: | Line 22: | ||
#Autoclave (liquid cycle) | #Autoclave (liquid cycle) | ||
#* 250°'''F''', 22psi, 30 minutes | #* 250°'''F''', 22psi, 30 minutes | ||
Notes: We do not pH medium when we make it on the fly. However, if it is really important, pH the medium to 7.0 with 5M NaOH (~200µL). Check with pH paper | |||
===For plates=== | |||
#Mix dry ingredients and add distilled water up to less than 1 Liter | |||
#After dissolved, add agar | |||
#Pour into 2 L flask (or greater) | |||
#Autoclave (liquid cycle) | |||
#* 250°'''F''', 22psi, 30 minutes | |||
#Cool media down to 60-55C (uncomfortable but not painful) | |||
#Add appropriate antibiotic | |||
#Pour the solution on sterile (???) plates | |||
#Let solidify about 1 h, wrap, label (name, date, additive) | |||
#keep at 4°C | |||
==Source== | ==Source== | ||
J. Sambrook, D.W. Russell, ''Molecular Cloning: A Laboratory Manual'' (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) | Adapted From: | ||
J. Sambrook, D.W. Russell, ''Molecular Cloning: A Laboratory Manual'' (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2 | |||
[[Category:Material]] [[Category:Standard Media]] | |||
Latest revision as of 06:56, 31 March 2015
Summary
Luria-Bertani Medium (aka L-Broth or LB Medium). (Bertani says LB really stands for lysogeny broth.) LB is a standard growth medium for a variety of bacteria and conditions.
LB contains a milk digest (tryptone) and hence can induce cultures with lac based promoters when they near saturation.
- "LB ... contains enzymatic digests of the milk protein casein (e.g., tryptone or N-Z-amine) and yeast extract. Since milk is rich in lactose, an inducer of the T7 expression system, variable amounts of residual lactose may be present in different lots of these enzymatic digests. These small amounts of lactose do not promote appreciable induction during log-phase growth, but even minute amounts are sufficient to cause induction on approach to saturation, particularly at lower rates of aeration, which allow induction at lower lactose concentration and promote higher levels of induction." -- Sturdier 2014
Ingredients
- 10 g Bacto-tryptone
- 5 g yeast extract
- 10 g NaCl
For plates also add
- 15g agar
See also: Silver: LB Liquid
Note: There are two formulations of LB, Miller and Lennox, that differ in the amount of NaCl. Lennox has less salt, only 5 g/L. The Qiagen miniprep kit recommends LB with 10 g NaCl for highest plasmid yields.
Protocol
- Mix dry ingredients and add distilled water up to 1 Liter
- Pour into 2 L flask (or greater)
- Autoclave (liquid cycle)
- 250°F, 22psi, 30 minutes
Notes: We do not pH medium when we make it on the fly. However, if it is really important, pH the medium to 7.0 with 5M NaOH (~200µL). Check with pH paper
For plates
- Mix dry ingredients and add distilled water up to less than 1 Liter
- After dissolved, add agar
- Pour into 2 L flask (or greater)
- Autoclave (liquid cycle)
- 250°F, 22psi, 30 minutes
- Cool media down to 60-55C (uncomfortable but not painful)
- Add appropriate antibiotic
- Pour the solution on sterile (???) plates
- Let solidify about 1 h, wrap, label (name, date, additive)
- keep at 4°C
Source
Adapted From:
J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2
