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<span style="font-size: 14pt; color: red; font-weight: bold;"><center> We're currently hard at work on the 3A Assembly Kit manual. We've added preliminary protocols for all of the necessary steps so you can read through them for now!</center></span>
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<img src="https://static.igem.org/mediawiki/2013hs/9/91/3A_Assembly_and_Transformation_Efficiency_kits.jpg" style="height: 300px; border: 1px solid #000;">
 
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You've just received the 3A Assembly Kit! We hope your team will enjoy using the kit and in the process learn about synthetic biology based on standard parts and 3A assembly. Before you start please make sure to...
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<b>Hi High Schoolers,</b>
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<p>You've just received the 3A Assembly Kit! We hope your team will enjoy using the kit and in the process learn about synthetic biology based on standard parts and 3A assembly. Before you start please make sure to...</p>
 
<ul>
 
<ul>
 
<li>check the contents of the box with the <a href="https://parts.igem.org/Help:3A_Assembly_Kit/Inventory">inventory</a>, to make sure you have everything</li>
 
<li>check the contents of the box with the <a href="https://parts.igem.org/Help:3A_Assembly_Kit/Inventory">inventory</a>, to make sure you have everything</li>
 
<li>inspect the materials to make sure nothing has been damaged during shipping</li>
 
<li>inspect the materials to make sure nothing has been damaged during shipping</li>
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<li>refrigerate the agar plates and any materials labeled as requiring storage at 4C.</li>
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<li>refrigerate the agar plates and any materials labeled as requiring storage at 4C. </li>
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<li>order the NEB reagent kit. You'll need to use the NEB enzymes and reagents for 3A assembly. See the <a href="http://2012hs.igem.org/Sponsors">High School Division sponsor page</a> for more information.</li>
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<li>order an NEB enzyme kit. Click <a href="https://www.neb.com/products/E0546-BioBrick-Assembly-Kit">here</a> for details.
 
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You can download a <a href="https://static.igem.org/mediawiki/parts/e/e9/3A-Assembly-Kit-HiRes.png">hi-res png</a> or <a href="https://static.igem.org/mediawiki/parts/0/0e/3A-Assembly-Kit.pdf">pdf</a> of the images in the protocol.
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If you're interested in using the 3A Assembly Kit, and would like to order one, please contact <strong>hq (at) igem (dot) org</strong>.
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===Introduction===
 
===Introduction===
  
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The 3A Assembly Kit and following protocols will take you through the process of 3A Assembly, and by the end you will have assembled your own composite part in the lab. The kit includes two parts: '''Part A''' (<partinfo>BBa_J04500</partinfo>) and '''Part B''' (<partinfo>BBa_J04650</partinfo>), which when assembled together will form a RFP (red fluorescent protein) generator. Your cells will turn <span style="color: red; font-weight: bold;">red!</span>  
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The 3A Assembly Kit and following protocols will take you through the process of [[Help:Assembly#3A_Assembly | 3A Assembly]], and by the end you will have assembled your own composite part in the lab. The kit includes two parts: '''Part A''' (<partinfo>BBa_J04500</partinfo>) and '''Part B''' (<partinfo>BBa_J04650</partinfo>), which when assembled together will form a RFP (red fluorescent protein) generator. Your cells will turn <span style="color: red; font-weight: bold;">red!</span>  
  
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'''Note:''' The 3A Assembly Kit also includes prepared plasmid DNA for '''Part A''' and '''Part B''', so you can skip directly to '''Step #3: Restriction Digest''' if you'd like.
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'''Note:''' The 3A Assembly Kit also includes prepared plasmid DNA for '''Part A''' and '''Part B''', so you can skip directly to '''Step #3: Restriction Digest''' if you'd like. But don't forget to take a look at '''Step #1: Grow,''' as you'll need to follow the instructions for growing up cells for '''Step #5: Transformation'''.
  
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===Protocols===
  
 
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<td class="headerstep" colspan=2><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Growing">Step #1: Growing</a></td>
 
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<p><strong>Grow up the E. coli that contains your parts!</strong> <br>Your kit includes agar stabs for Part A and Part B. The agar stabs contain live bacteria (E. coli) that house your parts. The parts themselves belong to a plasmid which replicates within the E. coli cell.</p>
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<p><strong>Grow up the E. coli that contains your parts!</strong></p>
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<p>Before working with your parts, you'll need to make sure you have enough copies of them. You can do this by growing up cell cultures from the agar stabs: more E.coli cells means more plasmids, which means more copies of your parts. Use the provided agar stabs to streak out the bacteria containing your parts onto agar plates. Then pick a single colony to grow up a cell culture.</p>
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<p><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Growing">Click here for the Growing Protocol</a></p>
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<p>The 3A Assembly Kit also includes an agar stab with the NEB 10-beta strain that you'll need to make competent cells for '''Step #5: Transformation.''' Please be sure to follow the growing instructions for the NEB 10-beta strain, as you'll need to prepare these two days in advance to '''Step #5: Transformation.'''</p>
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<p><strong>Miniprepping cell cultures to extract the DNA for your parts.</strong> <br>You now have cell cultures of E. coli containing lots of copies of Part A and Part B. In order to access the plasmid DNA containing your part, you'll need to lyse the cells (make them burst open), separate the DNA from the cellular material, and purify the sample to make sure you only have the plasmid DNA and not the E. coli's own genomic DNA. The 3A Assembly Kit includes the reagents and tubes required for the Miniprep step, however, <strong>a centrifuge is required</strong>.</p>
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<p><strong>Miniprep cell cultures to extract the DNA for your parts.</strong></p>
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<p>If you would simply like to skip the Miniprep step, the 3A Assembly Kit includes prepared plasmid DNA for '''Part A''' and '''Part B''', so you can skip directly to '''Step #3: Restriction Digest.'''
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<p><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Miniprep">Click here for the Miniprep Protocol</a></p>
 
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<strong>Cutting the DNA for your parts and the pSB1C3 linearized plasmid backbone in a restriction digest</strong> <br>Now that you have Part A and Part B, you'll want to cut them out from their pSB1AK3 plasmid backbones. You'll use restriction enzymes (provided by NEB) to cut the parts out. This will leave small overhangs at the end of each part which will eventually act like connectors to assembled them together.  
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<p><strong>Cut the DNA for your parts and the pSB1C3 linearized plasmid backbone in a restriction digest</strong></p>  
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<p><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Restriction_Digest">Click here for the Restriction Digest Protocol</a></p>
 
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<strong>Ligating your two parts together into the pSB1C3 linearized backbone</strong> <br>After your restriction digest you'll have Part A, Part B, and the pSB1C3 linearized plasmid backbone cut. Now you'll need to assemble all of these components together (Part A + Part B, into pSB1C3). You'll do this through the use of the overhangs (connectors) you've created by cutting the DNA with restriction enzymes and ligase, which will keep everything glued together.
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<p><strong>Ligate your two parts together into the pSB1C3 linearized backbone</strong></p>  
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<p><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Ligation">Click here for the Ligation Protocol</a></p>
 
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<td class="headerstep" colspan=2><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Transformations">Step #5: Transformation</a></td>
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<td class="headerstep" colspan=2><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Transformation">Step #5: Transformation</a></td>
 
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<strong>Transforming the ligation to get your newly assembled composite part. At the end you should see red colonies!</strong> <br>You now have an assembled product through ligation (Part A + Part B, in pSB1C3). To get this new part into E. coli cells, you'll need to use a process called transformation. You'll need to make the provided NEB 10-beta strain competent, so it can uptake your ligated product (the plasmid containing your new part). Once the cells have taken in your plasmid, you'll grow them on a chloramphenicol plate (only cells containing the pSB1C3 plasmid backbone will live), and from those cells that live, there will be red colonies that have the correct new part.
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<p><strong>Transform the ligation into E. coli to get your newly assembled composite part.</strong></p>
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<p><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Transformation">Click here for the Transformation Protocol</a></p>
 
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<td class="headerstep" colspan=2><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Results">Step #6: Results</a></td>
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<td class="headerstep" colspan=2><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Results">Post your Results!</a></td>
 
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<strong>Post your results!</strong> <br>Hopefully you've learned quite a bit while using the 3A Assembly Kit. Use the Results page to post what happened during your assembly.
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<p><strong>Post your results! Hopefully you've learned quite a bit while using the 3A Assembly Kit. </strong></p>
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<p><a href="https://parts.igem.org/Help:3A_Assembly_Kit/Results">Click here to post your Results!</a></p>
 
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[[Category:Protocols]]

Latest revision as of 16:51, 1 June 2017

This page has been discontinued. We apologize for any inconvenience. For more information please contact iGEM HQ

Reason: We are no longer providing the 3A Assembly Kit (as of the 2015 season).


The 3A Assembly Kit
Hi High Schoolers,

You've just received the 3A Assembly Kit! We hope your team will enjoy using the kit and in the process learn about synthetic biology based on standard parts and 3A assembly. Before you start please make sure to...

  • check the contents of the box with the inventory, to make sure you have everything
  • inspect the materials to make sure nothing has been damaged during shipping
  • refrigerate the agar plates and any materials labeled as requiring storage at 4C.
  • order an NEB enzyme kit. Click here for details.

You can download a hi-res png or pdf of the images in the protocol.

Introduction

The 3A Assembly Kit and following protocols will take you through the process of 3A Assembly, and by the end you will have assembled your own composite part in the lab. The kit includes two parts: Part A (BBa_J04500) and Part B (BBa_J04650), which when assembled together will form a RFP (red fluorescent protein) generator. Your cells will turn red!

Note: The 3A Assembly Kit also includes prepared plasmid DNA for Part A and Part B, so you can skip directly to Step #3: Restriction Digest if you'd like. But don't forget to take a look at Step #1: Grow, as you'll need to follow the instructions for growing up cells for Step #5: Transformation.

Protocols

Step #1: Growing

Grow up the E. coli that contains your parts!

Click here for the Growing Protocol
Step #2: Miniprep

Miniprep cell cultures to extract the DNA for your parts.

Click here for the Miniprep Protocol
Step #3: Restriction Digest

Cut the DNA for your parts and the pSB1C3 linearized plasmid backbone in a restriction digest

Click here for the Restriction Digest Protocol
Step #4: Ligation

Ligate your two parts together into the pSB1C3 linearized backbone

Click here for the Ligation Protocol
Step #5: Transformation

Transform the ligation into E. coli to get your newly assembled composite part.

Click here for the Transformation Protocol
Post your Results!

Post your results! Hopefully you've learned quite a bit while using the 3A Assembly Kit.

Click here to post your Results!