Team:Sumbawagen/Notebook/protocol8

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<h2>Notebook – Protocol – 8. Transformation </h2>
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<h2>Notebook – Protocol – 8. Transformation (Heat Shock)</h2
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<p><strong>A. Purpose</strong></p>
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<p>Since the amount of DNA that comes with the DNA distribution kit is not enough for assembly, we need to transform this DNA into cells and then make our own stocks with sufficient amount of DNA. The principle of this step is to put the plasmid that contain a small amount DNA of interest into competent cells, let them grow (replicate) and then harvest back the amplified plasmid that contain the gene of interest.</p>
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<p><strong>B. Protocol</strong></p>
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<p><strong>Transformation Method I</strong></p>
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<p>We used TSS method in this transformation process. The method created and developed by Chung, et al and the protocol was adapted in Dr. Arief Budi Witarto’s experiments.</p>
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<p><strong>Preparation</strong></p>
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<ul style="padding-left:25px;">
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<li> Equipments : Centrifuge, horizontal shaker, incubator, micropipette, 1,5 ml microtube, 15 ml tube.</li>
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<li> Materials : LB culture for culturing the bacteria, E. coli BL21(DE3) competent cells as the cell host, plasmid Bba_J04450 50 pg/ul as the vector, TSS medium, 1 M glucose, TSS medium.</li>
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<p>1. Pre-culture</p>
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<ul style="padding-left:25px;">
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<li> Culture the Escherichia coli BL21(DE3) competent cells by adding 100 µl of E. coli. BL21(DE3) glycerol stock into 3 ml of LB liquid medium.</li>
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<li> Incubate with shaking it at room temperature (28-20 °C) for 3 hours.</li></ul>
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<p>2. Plasmid transformation</p>
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<ul style="padding-left:25px;">
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<li> After 3 hours, add 1 ml of the pre-cultured results into 3 microtubes 1,5 ml steril.</li>
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<li> Pellets the cells by centrifugation at 4°C for 10 minutes.</li>
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<li> Discard as much as supernatant as possible.</li>
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<li> Mix the pellets in each tube by adding 1 ml TSS medium into the tubes. Suspend the pellets thoroughly with a micropipette. Suspended pellets then move into another tube that contain pellets, and suspend it again until we get 1 tube contain suspended<i> E. coli.</i></li>
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<li> Move 100 µl suspended <i> E. coli</i> into 2 cooled microtubes. (One tube using for control and another one for the transformation reaction).</li>
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<li> Add 2 µl plasmid/part BBa_J04450 50 pg/µl from part registry into 100 µl suspended<i> E. coli.</i></li>
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<li> Resuspency by pipetting and tapping the tube.</li>
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<li> Incubate the tubes for 5-6 hours at 4 °C.d<i> E. coli.</i></li>
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<li> After 5-6 hours, add 1 ml LB liquid medium that has been mixed with 18 µl of glucose 1 M.</li>
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<li> Incubate overnight with shaking in room temperature (28-30 °C).</li>
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</ul>
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<p>3. Plate out the cultured</p>
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<ul style="padding-left:25px;">
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<li> Pour 100 µl of the transformation results to agar plate containing appropriate antibiotic. We use CAM with 50 µg/µl final concentration in plate. </li>
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<li> Use spreader to spread evenly then incubate overnight at 37 °C .</li>
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<li> Centrifuge the remaining transformation results (900 µl) for 3 minutes</li>
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<li> Move 700 µl supernatant into empty tube.</li>
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<li> Pipetting the remaining supernatant with pellets (200 µl)</li>
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<li> Add 100 µl to LB plate containing appropriate antibiotic</i></li>
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</ul>
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<p><strong>Transformation Method II</strong></p>
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<p>The method used in this transformation process is heat shock method. The principle of this method is by giving high temperature to the cells. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell</p>
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<p><strong>Preparation</strong></p>
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<ul style="padding-left:25px;">
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<li> Equipments : Microcentrifuge, horizontal shaker, incubator, micropipette, tubes 15 ml, polipropylene tube (50 ml and 2 ml.</li>
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<li> Materials needed : LB liquid medium for culturing the bacteria, <i>E. coli</i> DH5-alpha competent cells as the cell host, plasmid Bba_J04450 as the vector. </li>
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<p>Steps</p>
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<p>Protocols from the kit were followed by adaptation because we have no high speed micro centrifuge, but only a 6,000 rpm small micro centrifuge </p>
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<ul style="padding-left:25px;">
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<li> Add plasmid Bba_J04450 (2 µl for 50 pg/µl, 5 µl for 20 pg/µl, etc) into 3 tube containing  <i>E. coli</i> DH5-alpha competent cell (tube 2A, 3A, and 4A) that have made before (see Protocol Making Competent Cell.</li>
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<li> Mix by pipetting using micropippet (5-10 times pipetting).</li></ul>
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<li> Incubate in refrigerator for 40 minutes.</li>
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<li> Prepare the hot water with the temperature of 42 °C, measure the temperature using thermometer.</li>
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<li> Heatshock the cells by put the bottom of the tube on hot water for  45 seconds.</li>
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<li> Incubate in refrigerator for 5 minutes. </i></li>
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<li> Add 1 ml LB liquid medium sterile (without antibiotic) into each tubes (tube 2A, 3A, and 4A). </li>
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<li> Shake for 2 hours.</li>
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<li> Plating the transformation by add 100 µl transformation reaction to LB agar plates containing chlorampenicol (final concentration 50 µg/ml).</li>
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<li> Label the plates with 2a-1x, 2b-1x, and 2c-1x.</i></li>
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<li> Centrifuge the remaining transformation results for 3 minutes.</li>
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<li> Discard as much as 700 µl supernatant</li>
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<li> Suspend the remaining supernatant and pellet by pippeting.</li>
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<li> Add 100 µl to agar plate containing chloramphenicol (final concentration 50 µg/µl).</li>
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<li> Label the plates with 2a-1x, 2b-1x, and 2c-1x.</i></li>
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<li> Incubate at 37 °C overnight.</li>
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<li> Check the result later.</li>
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</ul>
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<p>1.  Add plasmid into tubes containing E. coli competent cell.</p>
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<p>2.  Mix by pipetting using micropipette (5-10 times pipetting). </p>
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<p>3.  Incubate at 4 °C for 40 minutes (inside a refrigerator).</p>
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<p>4.  Prepare the hot water with the temperature of 42 °C, measure the temperature using thermometer. </p>
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<p>5.  Heat shock the cells by put the bottom of the tube on hot water for 45 seconds.</p>
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<p>6.  Incubate at 4 °C for 5 minutes.</p>
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<p>7.  Add 1 ml LB liquid medium sterile (without antibiotic) into each tubes.</p>
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<p>8.  Incubate using shaker for 2 hours</p>
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<p>9.  Plating the transformation by add 100 µl transformation reaction to LB agar plates containing chloramphenicol (final  concentration 50 µg/ml).</p>
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<p>10. Give label for the plates.</p>
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<p>11. Centrifuge for 6,000 rpm for 3 minutes.</p>
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<p>12. Discard the flow-through as much as 700 µl.</p>
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<p>13. Suspend the remaining flow-through and pellets by pipetting.</p>
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<p>14. Add 100 µl to agar plate containing appropriate antibiotic.</p>
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<p>15. Give label for the plates.</p>
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<p>16. Incubate at 37 °C overnight.</p>
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<p>17. Check the result later. </p>
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Latest revision as of 09:23, 19 February 2015

Team:Dundee/Team - 2013.igem.org

 

Team:Sumbawagen/Team

From 2014.igem.org

iGEM Sumbawagen 2014 · Econey

Sumbawagen

Notebook – Protocol – 8. Transformation (Heat Shock)

Protocols from the kit were followed by adaptation because we have no high speed micro centrifuge, but only a 6,000 rpm small micro centrifuge

1. Add plasmid into tubes containing E. coli competent cell.

2. Mix by pipetting using micropipette (5-10 times pipetting).

3. Incubate at 4 °C for 40 minutes (inside a refrigerator).

4. Prepare the hot water with the temperature of 42 °C, measure the temperature using thermometer.

5. Heat shock the cells by put the bottom of the tube on hot water for 45 seconds.

6. Incubate at 4 °C for 5 minutes.

7. Add 1 ml LB liquid medium sterile (without antibiotic) into each tubes.

8. Incubate using shaker for 2 hours

9. Plating the transformation by add 100 µl transformation reaction to LB agar plates containing chloramphenicol (final concentration 50 µg/ml).

10. Give label for the plates.

11. Centrifuge for 6,000 rpm for 3 minutes.

12. Discard the flow-through as much as 700 µl.

13. Suspend the remaining flow-through and pellets by pipetting.

14. Add 100 µl to agar plate containing appropriate antibiotic.

15. Give label for the plates.

16. Incubate at 37 °C overnight.

17. Check the result later.

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