Team:TU Darmstadt/protocols/Chemically competent cells

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!align="center"|[[Team:TU_Darmstadt/labbook|<span style="color:white;font-size:160%;"> Labbook |</span>]]
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!align="center"|[[Team:TU_Darmstadt/protocols|<span style="color:white;font-size:160%;"> Protocols |</span> ]]
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!align="center"|[[Team:TU_Darmstadt/materials|<span style="color:white;font-size:160%;"> Materials |</span>]]
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== SOC ==
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SOC medium is used in [https://2013.igem.org/Team:TU_Darmstadt/protocols/Heat_Shock_Transformation bacterial transformation].
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=== Ingredients ===
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<!-- Chemically competent cells -->
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*[https://2013.igem.org/Team:TU_Darmstadt/materials/SOB SOB]
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*20 mM glucose
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<h2><font size="6" color="#F0F8FF" face="Arial regular"> Chemically competent cells </font></h2>
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===Protocol===
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#Make 1 L of [https://2013.igem.org/Team:TU_Darmstadt/materials/SOB SOB] media
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#After autoclaving and cooling to less than 50°C, add 20 ml filter sterilized 20% glucose solution
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<B> Short explanation<br></B>
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The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.<br>
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<br>
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<B> Equipment <br></B>
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-80°C freezer<br>
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Incubation shaker<br>
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Centrifuge (cooling cababilities required!)<br>
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photometer<br>
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Ice water bath<br>
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<br>
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<B> Chemicals & consumables <br></B>
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Ice and/or liquid nitrogen<br>
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Falcon tubes<br>
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Eppis<br>
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dYT Medium (50 ml p.c.)<br>
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ice cold 100mM CaCl2<br>
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Glycerin<br>
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<br>
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<B> Procedure<br></B>
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1. Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight<br>
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2. Inoculate 200 mL LB with the preculture <br>
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3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached<br>
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4. Incubate cells on ice for 15 min<br>
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5. Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice)<br>
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6. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!)<br>
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7. Incubate on ice for 1 hour<br>
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8. Centrifuge the culture at 4°C and 3000 x g for 10 min<br>
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9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2<br>
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10. Incubate on ice for 1 hour<br>
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11. Centrifuge the culture at 4°C and 3000 x g for 5 min<br>
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12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine<br>
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13. Incubate on ice for 30 min<br>
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14. Aliquot the cells à 100µL<br>
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15. Store at -80°C<br>
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<br>

Revision as of 17:36, 4 October 2013





Chemically competent cells

Short explanation
 The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.

Equipment
-80°C freezer
Incubation shaker
Centrifuge (cooling cababilities required!)
photometer
Ice water bath

Chemicals & consumables
Ice and/or liquid nitrogen
Falcon tubes
Eppis
dYT Medium (50 ml p.c.)
ice cold 100mM CaCl2
Glycerin

Procedure
 1. Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight
2. Inoculate 200 mL LB with the preculture
3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached
4. Incubate cells on ice for 15 min
5. Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice)
6. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!)
7. Incubate on ice for 1 hour
8. Centrifuge the culture at 4°C and 3000 x g for 10 min
9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2
10. Incubate on ice for 1 hour
11. Centrifuge the culture at 4°C and 3000 x g for 5 min
12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine
13. Incubate on ice for 30 min
14. Aliquot the cells à 100µL
15. Store at -80°C

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/protocols/Chemically_competent_cells"