Team:Paris Bettencourt/Protocols

From 2013.igem.org

(Difference between revisions)
(→Protocol 2: CaCl2 Competent Cells)
Line 62: Line 62:
</body>
</body>
</html>
</html>
 +
 +
== <b> Protocol 4: </b> E.coli Chromosomal Integration ==
 +
<html>
 +
<body>
 +
<ol>
 +
<b>Protocol: <i>E. coli</i> Chromosomal Integration</b> (&agrave; la Datsenko and Wanner)<br />
 +
<br />
 +
<b>Preparation of Electrocompetent Cells</b><br />
 +
Note: Competent cells should never be vortexted, as this will cause them to lyse <br />
 +
and release salts into the media. Resuspend cells by pipeting up and down with a large <br />
 +
pasteur pipet. Once they are chilled, cells should be continuously cold.<br />
 +
<br />
 +
<li>The night before the transformation, start an overnight culture of cells.<br />
 +
5 ml LB Amp.<br />
 +
<br />
 +
<li>The day of the transformation, dilute the cells 100X.<br />
 +
100 ml LB Amp.<br />
 +
Grow at 30&deg;C for about 90 minutes.<br />
 +
<br />
 +
<li>Harvest the cells.<br />
 +
When the cells reach an OD600 of between 0.6 and 0.8.<br />
 +
Split the culture into 2x 50 ml falcon tubes, on ice.<br />
 +
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br />
 +
<br />
 +
<li>Wash and combine the cells.<br />
 +
Remove the supernatant.<br />
 +
Resuspend the cells in 2x 25 ml of ice cold water.<br />
 +
Combine the volumes in a single 50 ml falcon tube.<br />
 +
<br />
 +
<li>Wash the cells 2 more times.<br />
 +
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br />
 +
Resuspend in 50 ml of ice cold water.<br />
 +
Repeat.<br />
 +
<br />
 +
<li>Wash and concentrate the cells for electroporation.<br />
 +
Centrifuge at 4 &deg;C for 10 min at 4000 rpm.<br />
 +
Resuspend in 1-2 ml of ice cold water.<br />
 +
We will use 200 ul of washed cells per transformation.<br />
 +
</ol>
 +
<ol>
 +
<br />
 +
<b>Dialysis of PCR or Digestion Products</b><br />
 +
Note: DNA for electroporation must be free of salts to avoid arcing.<br />
 +
<br />
 +
<li>Float a filter in a Petri dish filled with water.<br />
 +
Millipore membrane filter 0.025 uM.<br />
 +
<br />
 +
<li>Pipet one drop of PCR product onto the filter.<br />
 +
200 ng is needed per transformation.<br />
 +
20 - 100 ul fits well on one filter.<br />
 +
<br />
 +
<li>Collect the drop after 30 - 45 minutes.<br />
 +
The volume will change, but the DNA is not lost.
 +
</ol>
 +
</body>
 +
</html>
 +
<!-- ########## Don't edit below ########## -->
<!-- ########## Don't edit below ########## -->
{{:Team:Paris_Bettencourt/footer}}
{{:Team:Paris_Bettencourt/footer}}

Revision as of 12:50, 23 July 2013

<body>

Protocols

Contents

Protocol 1: Heat Shock Transformation

  1. Thaw 20 ul of Chemically Competent Cells on Ice
  2. Add 2 ul DNA (intact plasmid)
  3. Incubate on Ice for 30 minutes
  4. Incubate at 42C for 45 seconds
  5. Incubate on Ice for 2 minutes
  6. Add 200 ul of LB broth
  7. Incubate at 37C for 1 hour in shaker
  8. Plate on Agar supplemented with appropriate antibiotics.

Protocol 2: CaCl2 Competent Cells

This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.

Note: Never vortex competent cells.

Resuspend by pipetting with large Pasteur pipettes.

    The night before:

  1. The night before, inoculate a 5 ml culture and grow overnight with selection.

    The day of:

  2. Dilute cells ~ 1:200 into selective media.
    For this example add 250 ul to 50 ml of selective media.
    Note: The protocol is easily scaled to increase the number of cells.
  3. Grow the cells to an OD600 of 0.6 – 0.7.
    Use a large flask, 500ml, for good aeration.
    Use a baffled flask for fastest growth.
    This takes about 3 hours depending on the cells.
    Medium-heavy cloudiness by eye is fine.
  4. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
  5. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
  6. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
  7. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
  8. Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
Note: Frozen cells are only good once.Do not refreeze cells once thawed.

Protocol 3: Glycerol Stocks

  1. Pick Single colonies from agar plates
  2. Innoculate 5ml LB broth overnight.
  3. Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.
  4. Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.

Protocol 4: E.coli Chromosomal Integration

    Protocol: E. coli Chromosomal Integration (à la Datsenko and Wanner)

    Preparation of Electrocompetent Cells
    Note: Competent cells should never be vortexted, as this will cause them to lyse
    and release salts into the media. Resuspend cells by pipeting up and down with a large
    pasteur pipet. Once they are chilled, cells should be continuously cold.

  1. The night before the transformation, start an overnight culture of cells.
    5 ml LB Amp.

  2. The day of the transformation, dilute the cells 100X.
    100 ml LB Amp.
    Grow at 30°C for about 90 minutes.

  3. Harvest the cells.
    When the cells reach an OD600 of between 0.6 and 0.8.
    Split the culture into 2x 50 ml falcon tubes, on ice.
    Centrifuge at 4 °C for 10 min at 4000 rpm.

  4. Wash and combine the cells.
    Remove the supernatant.
    Resuspend the cells in 2x 25 ml of ice cold water.
    Combine the volumes in a single 50 ml falcon tube.

  5. Wash the cells 2 more times.
    Centrifuge at 4 °C for 10 min at 4000 rpm.
    Resuspend in 50 ml of ice cold water.
    Repeat.

  6. Wash and concentrate the cells for electroporation.
    Centrifuge at 4 °C for 10 min at 4000 rpm.
    Resuspend in 1-2 ml of ice cold water.
    We will use 200 ul of washed cells per transformation.

    Dialysis of PCR or Digestion Products
    Note: DNA for electroporation must be free of salts to avoid arcing.

  1. Float a filter in a Petri dish filled with water.
    Millipore membrane filter 0.025 uM.

  2. Pipet one drop of PCR product onto the filter.
    200 ng is needed per transformation.
    20 - 100 ul fits well on one filter.

  3. Collect the drop after 30 - 45 minutes.
    The volume will change, but the DNA is not lost.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
team2013@igem-paris.org
Hit Counter by Digits
Copyright (c) 2013 igem.org. All rights reserved.

Retrieved from "http://2013.igem.org/Team:Paris_Bettencourt/Protocols"