Team:StanfordBrownSpelman/Lab Techniques2

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<b>Producing Plates</b><br>
<b>Producing Plates</b><br>
-
Using any typical media recipe, add 1.5% agar (15g/L) to the mixture before autoclaving
+
Using any typical media recipe, add 1.5% agar (15g/L) to the mixture before autoclaving. After removing from autoclave, let cool until it is cool enough to hold for several seconds comfortably (otherwise the media will be too hot and break down the antibiotic). Add the appropriate amount of antibiotic. Pour enough media into each petri dish to just cover the bottom. <i>E. coli</i> grows on the surface, so the agar layer shouldn’t be thickSince the dishes come in sleeves of 25, it is usually good to make 500mL of the medium
-
After removing from autoclave, let cool until it is cool enough to hold for several seconds comfortably (otherwise the media will be too hot and break down the antibiotic)
+
-
Add the appropriate amount of antibiotic
+
-
Pour enough media into each petri dish to just cover the bottom
+
-
E. coli grows on the surface, so the agar layer shouldn’t be thickSince the dishes come in sleeves of 25, it is usually good to make 500mL of the medium
+
-
<br />Keeping Them Sterile<br />
+
-
We have a UV hood that we usually make plates in
+
-
Gather everything you'll need to make the plates (empty petri dishes, pipette, pipette tip, sharpie, etc) and wipe down with 70% ethanol before placing in the hood
+
-
Sterilize for ~5-10min by exposing to UV lamp.
+
-
<br /><b>Liquid Culture</b>
+
<br /><br /><b>Keeping Them Sterile</b><br />
-
<br />Inoculation:
+
We have a UV hood that we usually make plates in. Gather everything you'll need to make the plates (empty petri dishes, pipette, pipette tip, sharpie, etc) and wipe down with 70% ethanol before placing in the hood. Sterilize for ~5-10min by exposing to UV lamp.
-
Pipette tips work great for swiping or stabbing a colony
+
-
<br />Media:
+
-
LB works great for both E. coli and B. subtilis
+
-
<br />Temperature:
+
-
37°C works fine for both E. coli and B. subtilis
+
-
<br />Shaker speed:
+
-
250 RPM for optimal growth, 200 OK
+
-
<br />Antibiotics:
+
-
If it is appropriate to select for the strain using antibiotics, add 1μl per mL of 1000X stock solution
+
-
<br />For best results (but certainly not necessary):
+
-
Pre-culture for ~6hrs in 20-25% final culture volume
+
-
Incubate in container with capacity >200% culture volume, overnight
+
-
<br /><b>Cryostocking</b>
+
<br /><br /><b>Liquid Culture</b>
 +
<br />Inoculation: Pipette tips work great for swiping or stabbing a colony
 +
<br />Media: LB works great for both E. coli and B. subtilis
 +
<br />Temperature: 37°C works fine for both E. coli and B. subtilis
 +
<br />Shaker speed: 250 RPM for optimal growth, 200 OK
 +
<br />Antibiotics: If it is appropriate to select for the strain using antibiotics, add 1μl per mL of 1000X stock solution
 +
<br />For best results (but certainly not necessary): Pre-culture for ~6hrs in 20-25% final culture volume. Incubate in container with capacity >200% culture volume, overnight
 +
 
 +
<br /><br /><b>Cryostocking</b>
<br />Any time you generate a new strain (i.e. transform a new combination of DNA parts) you
<br />Any time you generate a new strain (i.e. transform a new combination of DNA parts) you
should generate miniprep (for DNA) and a cryostock (for frozen cells).
should generate miniprep (for DNA) and a cryostock (for frozen cells).
-
<br /><br />Procedure
+
</h6>
-
<br />In a cryostock tube (1.8mL tube with screw top), mix a dense liquid culture of the strain with glycerol to the proper percentage (I think there's some flexibility but Jesse usually goes for 20% glycerol for both E. coli and B. subtilis)
+
<h6 class="subindent">
-
<br />(So this might look something like: 500μl liquid culture + 500μl 40% glycerol solution)
+
<br /><b>Procedure</b>
-
<br />Sterile technique is super important when making cryostocks!
+
<br />In a cryostock tube (1.8mL tube with screw top), mix a dense liquid culture of the strain with glycerol to the proper percentage (I think there's some flexibility but Jesse usually goes for 20% glycerol for both E. coli and B. subtilis) [So this might look something like: 500μl liquid culture + 500μl 40% glycerol solution]. Sterile technique is super important when making cryostocks!
</h6>
</h6>
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Latest revision as of 02:49, 16 October 2014

Stanford–Brown–Spelman iGEM 2014 — Amberless Hell Cell

Going Further
Producing Plates
Using any typical media recipe, add 1.5% agar (15g/L) to the mixture before autoclaving. After removing from autoclave, let cool until it is cool enough to hold for several seconds comfortably (otherwise the media will be too hot and break down the antibiotic). Add the appropriate amount of antibiotic. Pour enough media into each petri dish to just cover the bottom. E. coli grows on the surface, so the agar layer shouldn’t be thickSince the dishes come in sleeves of 25, it is usually good to make 500mL of the medium

Keeping Them Sterile
We have a UV hood that we usually make plates in. Gather everything you'll need to make the plates (empty petri dishes, pipette, pipette tip, sharpie, etc) and wipe down with 70% ethanol before placing in the hood. Sterilize for ~5-10min by exposing to UV lamp.

Liquid Culture
Inoculation: Pipette tips work great for swiping or stabbing a colony
Media: LB works great for both E. coli and B. subtilis
Temperature: 37°C works fine for both E. coli and B. subtilis
Shaker speed: 250 RPM for optimal growth, 200 OK
Antibiotics: If it is appropriate to select for the strain using antibiotics, add 1μl per mL of 1000X stock solution
For best results (but certainly not necessary): Pre-culture for ~6hrs in 20-25% final culture volume. Incubate in container with capacity >200% culture volume, overnight

Cryostocking
Any time you generate a new strain (i.e. transform a new combination of DNA parts) you should generate miniprep (for DNA) and a cryostock (for frozen cells).

Procedure
In a cryostock tube (1.8mL tube with screw top), mix a dense liquid culture of the strain with glycerol to the proper percentage (I think there's some flexibility but Jesse usually goes for 20% glycerol for both E. coli and B. subtilis) [So this might look something like: 500μl liquid culture + 500μl 40% glycerol solution]. Sterile technique is super important when making cryostocks!
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