Team:Cambridge/Protocols/Glycerol Stocks

From 2011.igem.org

(Difference between revisions)
(→Glycerol Stocks)
(→Glycerol Stocks)
Line 3: Line 3:
==Glycerol Stocks==
==Glycerol Stocks==
-
protocol description
+
Preparation of glycerol stocks of bacteria allows for long-term storage at -80°C without compromising viability of cells.
-
 
+
-
1. Peparation of liqid cultures  - 100-fold dilution of overnight culture with selection.
+
-
2. Incubation at 37° for 3-4 hours until mid-log phase.
+
-
3. Mix with 60% glycerol to get a final concentration of 20%.
+
-
4. Store at -80°
+
===Theory===
===Theory===
How it works
How it works
 +
===Practice===
===Practice===
-
How to do it in the lab
+
*'''''Preparation of glycerol stocks of bacteria'''''
-
 
+
:# Prepare a liquid culture of bacteria in LB with antibiotic for selection. Use 100-fold dilution of an overnight culture of the strain of interest.
-
{Standard layout for procedures is to use: 
+
:# Incubate cells at 37° for 3-4 hours until the culture reaches the mid-log phase.
-
 
+
:# Transfer around 1ml of the culture into an Eppendorf tube and mix with an appropriate amount of glycerol to get a final concentration of 20%.
-
*'''''<Procedure title - aka what you are doing>'''''
+
:# For high viabilitystore at -80&deg;.
-
:# <step 1>
+
-
:# <step 2>
+
:#* '''<additional notes/important information regarding the previous step>'''
:#* '''<additional notes/important information regarding the previous step>'''
-
the text within the < > is what should be written, don't include < > in actual writeup :P
+
'''Tip:''' When recovering a stored strain, it is advisable to check that the antibiotic markers have not been lost by
-
 
+
streaking the strain onto an LB-agar plate containing the appropriate antibiotic(s).
-
if in doubt see the gel electrophoresis protocol
+
'''Tip:'''Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria.
-
 
+
'''Tip:'''For precious strains, storage of two stock vials is recommended
-
}
+
===Safety===
===Safety===
-
The safety implication of the procedure.
+
All materials that come into contact with transgenic bacteria must be autoclaved.
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Revision as of 08:07, 21 September 2011

Loading...
OVERVIEW
home


Glycerol Stocks

Preparation of glycerol stocks of bacteria allows for long-term storage at -80°C without compromising viability of cells.

Theory

How it works

Practice

  • Preparation of glycerol stocks of bacteria
  1. Prepare a liquid culture of bacteria in LB with antibiotic for selection. Use 100-fold dilution of an overnight culture of the strain of interest.
  2. Incubate cells at 37° for 3-4 hours until the culture reaches the mid-log phase.
  3. Transfer around 1ml of the culture into an Eppendorf tube and mix with an appropriate amount of glycerol to get a final concentration of 20%.
  4. For high viabilitystore at -80°.
    • <additional notes/important information regarding the previous step>

Tip: When recovering a stored strain, it is advisable to check that the antibiotic markers have not been lost by streaking the strain onto an LB-agar plate containing the appropriate antibiotic(s). Tip:Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. Tip:For precious strains, storage of two stock vials is recommended

Safety

All materials that come into contact with transgenic bacteria must be autoclaved.


Retrieved from "http://2011.igem.org/Team:Cambridge/Protocols/Glycerol_Stocks"