McClean: Frogging a Serial Dilution

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: ==Overview== Frogging a serial dilution onto solid media is an effective way to distinguish and compare viability under certain growth conditions, etc. The serial dilution helps to elimina...)
Current revision (12:37, 20 May 2013) (view source)
 
Line 3: Line 3:
==Materials==  
==Materials==  
* 96-well plate (flat bottom wells)
* 96-well plate (flat bottom wells)
-
* "Frogger"
+
* Frogger
* Sterile petri dish about half-full of 95% ethanol
* Sterile petri dish about half-full of 95% ethanol
-
* Sterile petri dish
+
* Sterile petri dish about half-full of sterile water
-
 
+
* Multi-channel pipettor preferably with a range of 20 to 200 μL
-
==Stock Solutions==
+
-
 
+
-
'''Stock Solution 1'''
+
-
* This is a very simple solution, so we only need a one line description of how to make it.   
+
-
 
+
-
'''Stock Solution 2'''
+
-
 
+
-
This is a more involved solution, so we will describe how to make it in several steps:
+
-
# Step 1
+
-
# Step 2
+
-
# Step 3
+
==Protocol==
==Protocol==
-
# Step 1
+
# Take your cells directly from culture (can be overnight or subculture) and load 200 μL of each sample into a well in the first column (left-most is conventional)of the 96 well plate.
-
# Step 2
+
# Fill the rest of the wells in the rows that you are using with 180 μL sterile water.
-
# Step 3
+
# Take the multi-channel pipettor, set it to 20 μL, pipette the cells in the first column of wells up and down a few times to suspend, take 20 μL from these wells and deposit it in the adjacent (to the right) column of cells. Pipette up and down to suspend.
-
 
+
# After you pipette to mix the cells, take 20 μL from this set of wells and do the same for the next. Continue until you reach the last column of wells on the plate.
 +
# Sterilize the frogger by dipping the prongs into EtOH. After a quick shake insert the prongs into a Bunsen burner flame. Be CAREFUL not to burn yourself. Allow the frogger to cool slightly (1 minute). Alternatively, you can dip the frogger in sterile water for about 10 to 15 seconds to cool. Be sure to shake off the excess water.  If  you do not allow the frogger to cool enough, you will kill your cells and the frogging won't work well.
 +
#Align the prongs of the frogger with the wells and then place the prongs into the wells. Wiggle the frogger within the well to ensure that the culture in each well is uniformly mixed. Stamp frogger firmly but gently onto an agar plate. Repeat this step to stamp additional plates.
==Notes==
==Notes==
-
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
 
-
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
 
-
#List troubleshooting tips here. 
 
-
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
 
-
#Anecdotal observations that might be of use to others can also be posted here. 
 
-
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
 
-
==References==
 
-
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS
 
-
CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
 
==Contact==
==Contact==
<!--Change the information below to your info if you add a new protocol-->
<!--Change the information below to your info if you add a new protocol-->
-
*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 20 July 2011 (EDT)'''
+
*'''[[User:Michael T. Patel|Michael T. Patel]] 12:37, 20 May 2013 (EDT)''':or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
-
 
+
-
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
+
<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
Line 48: Line 28:
<!-- Move the relevant categories above this line to tag your protocol with the label
<!-- Move the relevant categories above this line to tag your protocol with the label
 +
 +
[[Category:Protocol]]
[[Category:Protocol]]

Current revision

Contents

Overview

Frogging a serial dilution onto solid media is an effective way to distinguish and compare viability under certain growth conditions, etc. The serial dilution helps to eliminate the possibility that a relatively high cell concentration could be misinterpreted as healthy growth, for example.

Materials

  • 96-well plate (flat bottom wells)
  • Frogger
  • Sterile petri dish about half-full of 95% ethanol
  • Sterile petri dish about half-full of sterile water
  • Multi-channel pipettor preferably with a range of 20 to 200 μL

Protocol

  1. Take your cells directly from culture (can be overnight or subculture) and load 200 μL of each sample into a well in the first column (left-most is conventional)of the 96 well plate.
  2. Fill the rest of the wells in the rows that you are using with 180 μL sterile water.
  3. Take the multi-channel pipettor, set it to 20 μL, pipette the cells in the first column of wells up and down a few times to suspend, take 20 μL from these wells and deposit it in the adjacent (to the right) column of cells. Pipette up and down to suspend.
  4. After you pipette to mix the cells, take 20 μL from this set of wells and do the same for the next. Continue until you reach the last column of wells on the plate.
  5. Sterilize the frogger by dipping the prongs into EtOH. After a quick shake insert the prongs into a Bunsen burner flame. Be CAREFUL not to burn yourself. Allow the frogger to cool slightly (1 minute). Alternatively, you can dip the frogger in sterile water for about 10 to 15 seconds to cool. Be sure to shake off the excess water. If you do not allow the frogger to cool enough, you will kill your cells and the frogging won't work well.
  6. Align the prongs of the frogger with the wells and then place the prongs into the wells. Wiggle the frogger within the well to ensure that the culture in each well is uniformly mixed. Stamp frogger firmly but gently onto an agar plate. Repeat this step to stamp additional plates.

Notes

Contact

Personal tools