View source for Team:Birkbeck/Electro-Competent E. coli Cell Protocol

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<h2><b><u>Creating electrocompetent cells</u></b></h2>
<h1>Making of competent cells</h1> <!-- – protocol


Materials
Conical flask – 2 L (autoclaved)
Falcon tubes
Bunsen burner
Pipettes (various)
Pipette tips
0.2 microtubes
1x plastic bag
Marker pen
Polystyrene box for ice/dry ice
Beaker for liquid waste
Corning® Filter System

Equipment
Fridge (4°C)
Freezer (-80°C)
Refrigerated Centrifuge for 50 mL Falcon tubes
Vacuum pump
Incubator (37 and 30°C) with shaking

Reagents
LB media (autoclaved)
10-β E. coli cells (thawed from frozen at -80C)
Glycerol [50% and filter-sterilised ideally]
Dry ice
Hepes buffer pH 7.0 solution 1 M (Sigma)
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<p><b><u><!--Step 2 – -->


Step 1 - Overnight Culture Preparation</u></b></p><!--Step 1 – , in the afternoon / evening-->
<!--Prepare in aseptic conditions(using a Bunsen burner)-->
<ul>
	<li>1.	Transfer 10 mL of sterile LB to a 50 mL Falcon Tube. </li>
	<li>2.	Inoculate media with a single colony of <i>E. coli</i> cells.</li>
	<!--<li>3.	Add a single colony or scrap of frozen glycerol stock or small aliquot of liquid culture of 10-β E. coli cells.</li>
4.	DO NOT ADD ANTIBIOTIC!!!-->
	<li>3.	Incubate overnight at 37°C, in a shaker at 230 rpm.</li>

<p><b><u><!--Step 2 – -->Step 2 - Preparing Electrocompetent <i>E. coli</i> Cells for Storage.<!--, in the morning--></u></b></p>
<ul>
<!--(<p><b>Preparing in aseptic conditions (using a Bunsen burner)--></b></p>
	<li>1.	Add 150-200 mL of LB media to the autoclaved 2 L conical (NB/ culture volume must be 1/10 of the total flask volume).</li>
	<li>2.	Use a 0.5% inoculum from step 1.</li>
	<li>3.	Incubate at 30°C until OD<sub>600</sub> ~0.6 (approximately 4 hours).</li>
	<li>4.	When culture reaches OD<sub>600</sub> = 0.6, cool to 4°C</li>
</ul>

<p><b><u><!--Step 3 – -->Step 3 (after culture OD<sub>600</sub> ~ 0.6 has been achieved)<!--, afternoon --></u></b></p>
<!--<ul>
	<li>1.	Prepare 1 mM Hepes buffer solution (in dH2O)</li>
	<li>2.	Filter Sterilise the solution</li>
	<li>3.	Cool to to 4°C</li>
</ul>
<p><b><u>Step 4 – Day 2, afternoon</u></b></p>-->
<ul>
	<li>1.	Aliquot 50 mL of culture into Falcon tubes. Incubate on ice</li>
	<li>2.	Pre-cool centrifuge to 4°C</li>
	<li>3.	Centrifuge the tubes for 10 mins at 4°C at 4000 rpm.</li>
	<li>4.	Decant supernatant.</li>
	<li>5.	Wash 1: resuspend pellet in 10 mL of 1 mM Hepes buffer (pH 7). Incubate on ice for 5 mins. Gently disturb tube every 30 seconds to facilitate resuspension of the pellet.<!--and intermittently shake the tubes, i.e. 0.5-1 min (keep them on ice as much as possible), to re-suspend the cells in the buffer (may take some time, i.e. 10-15 mins). Top up the tubes to 50 mL with 1 mM Hepes buffer--></li>
	<li>6.	Centrifuge for 10 mins at 4°C at 4000 rpm</li>
	<li>7.	Decant supernatant & resuspend pellet in 10 mL of 1 mM Hepes buffer (pH 7). Incubate on ice for 5 minutes. Gently disturb tube every 30 seconds to facilitate resuspension of the pellet. </li>
	<li>8.	Add two of the Falcon tubes together when cells are fully resuspended (giving two 20 mL cell suspensions). Top each cell suspension up to 50 mL with 1 mM Hepes (pH 7).</li>
	<li>9.	centrifuge again for 10 mins at 4°C at 4000 rpm.</li></li>
	<li>10.	Decant the supernatant & resuspend the pellet in 10 mL Hepes (pH 7). Incubate on ice for 5 minutes.</li>
	<li>11.	centrifuge again for 10 mins at 4°C at 4000 rpm.</li>
	<li>12.	Decant the supernatant & resuspend the pellet in 2 mL of Hepes (pH 7). Incubate on ice for 5 minutes.</li>
	<li>13.	Add glycerol to a final concentration of 12.5% (vol/vol). Mix thoroughly (but <em>GENTLY</em>) & incubate on ice for 5 minutes.</li>
	<li>14.	Aliquot 50 µL of cells suspension into 500 µL tubes and store at -80<sup>o</sup>C. </li>
</uL>
<br>
<li><a href="https://2015.igem.org/Team:Birkbeck/Experiments">Back to experiments and protocols</a></li>


<!--9.	Wash 2: Add 5-10 mL of 1 mM Hepes buffer. Place on ice for 5 mins. Gently and intermittently shake the tubes, i.e. 0.5-1 min (keep them on ice as much as possible), to re-suspend the cells in the buffer (may take some time, i.e. 10-15 mins). Join fractions (i.e. 4x 50 mL tubes  2x 50 mL tubes). Top up the tubes to 50 mL with 1 mM Hepes buffer and </li>
10.	centrifuge again for 10 mins at 4°C at 4000 rpm.</li>
11.	Pour out supernatant (cells will be in the pellet)</li>
12.	Wash 3: Add 5-10 mL of 1 mM Hepes buffer. Place on ice for 5 mins. Gently and intermittently shake the tubes, i.e. 0.5-1 min (keep them on ice as much as possible), to re-suspend the cells in the buffer (may take some time, i.e. 10-15 mins). Join fractions (i.e. 2x 50 mL tubes  1x 50 mL tubes). Top up the tube to 50 mL with 1 mM Hepes buffer and </li>
13.	centrifuge again for 10 mins at 4°C at 4000 rpm.</li>
14.	Pour out supernatant (cells will be in the pellet)</li>
15.	Add 1 mM Hepes buffer to produce final volume of 2 mL. Gently and intermittently shake the tubes, i.e. 0.5-1 min (keep them on ice as much as possible), to re-suspend the cells in the buffer (may take some time, i.e. 10-15 mins).</li>
16.	Add glycerol to 10-20% final concentration (usually 12.5%); mix gently for 2-3 mins, every 0.5-1 min.</li>
17.	Precool 0.5 microtubes on dry ice.</li>
18.	Aliquot 50 µL of cells to each 0.5 microtube (this should give ~40 aliquots)</li>
19.	Label a plastic bag, place all tubes into this plastic bag, transfer cells from dry ice directly to -80°C freezer.</li>
</ul>

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