Team:Macquarie Australia/Protocols/GibsonAssembly

From 2013.igem.org

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<h1>Gibson Assembly Protocol</h1>
<h1>Gibson Assembly Protocol</h1>
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Before entering the lab, the assigned Gibson Assembly team for the day organised agar plate requirements, concentration calculations and equipment requirements. All gBlocks had arrived and thus Gibson Assembly was performed on all fragments.<br><br>
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<center> <h7>
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Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on <b>NEB guidelines</b>. </center>
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<br><br>
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Each tube was labelled with antibiotic (C = Chloramphenicol, K = Kanamycin, A = Ampicillin) resistance contained within the allocated plasmid. Tubes labeled 1 & 2 represent Heme Oxygenase fragment mixtures, tubes labeled 3 represent Deinococcus fragment mixtures &  tubes labeled 4 & 5 represent Agrobacterium fragment mixtures. Take note that #1C and #4C contain T7 promoter fragments whereas the other BioBricks are T7 'promoter-less'. Deinococcus BioBrick does not contain any T7 promoters as described </html>[https://2012.igem.org/Team:Macquarie_Australia/Protocols/ArrivalofGBlocks here.]
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<b>NOTE:</b> For efficiency with reference to the NEB Gibson protocol, we considered:
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<br><br>
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<font size=3><b>1)</b></font> 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
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<font size=3><b>2)</b></font>  0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.
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Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer. The table below shows each tube with vector & volume, volume of Gibson Master Mix, water volume & volume of each gBlock fragment. After incubation, these were transformed into Top10 cells using the Transformation Protocol found here - 
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<br><br>
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</html>[[Team:Macquarie/Protocols/TransformationProtocol|Transformation Protocol]]. <html></p></html>
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A calculation provided was used furthermore to determine the pmols required based on the length of fragments: <br>
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<b>pmols</b> = (weight in ng) x 1,000 / (base pairs x 650 daltons)
<br><br>
<br><br>
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{| border="3" cellpadding="4" cellspacing="0" align="center" style="width: 100%; height: 400px"
 
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|-
 
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! scope="col" colspan="1" style="background-color: gray;"|
 
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! scope="col"| #1C
 
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! scope="col"| #2K
 
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! scope="col"| #2A
 
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! scope="col"| #3K
 
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! scope="col"| #3A
 
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! scope="col"| #4C
 
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! scope="col"| #5K
 
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! scope="col"| #5A
 
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|-
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<table border="0" cellpadding="10">
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! scope="row" rowspan="5" width="20%" | 1. Addition of 20ng Gene Block Fragments in appropriate tube
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<tr>
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| 2µl Hemo_T7A
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| 2µl Hemo_A
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| 2µl Hemo_A
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| 2µl Deino_A
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| 2µl Deino_A
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| 2µl Agro_T7A
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| 2µl Agro_A
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| 2µl Agro_A
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|-
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<td><img src="https://static.igem.org/mediawiki/2013/d/d6/Gibson_protocol.jpg" width=550 height=336></td>
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|  2µl Hemo_B
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<td>
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|  2µl Hemo_B
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|  2µl Hemo_B
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| 2µl Deino_B
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| 2µl Deino_B
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|  2µl Agro_B
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|  2µl Agro_B
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|  2µl Agro_B
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|-
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<center><font size=3><b>Legend</b></font></center>
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| nil
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| nil
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| nil
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| 2µl Deino_C
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| 2µl Deino_C
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|  2µl Agro_C
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|  2µl Agro_C
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|  2µl Agro_C
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<br>
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<font size=5>*</font> =      50ng of 5000 bp dsDNA is approx 0.015 pmols.
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<br><br>
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<font size=5>**</font> =  Control reagents
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<br><br>
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<font size=5>***</font> =  Additional master mix may be required for larger bp fragments.
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<br><br>
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<b> Note - </b>  50ng of 500 bp is approx 0.15 pmol
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<br><br>
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<b> Note - </b>  50-100ng of vector recommended with excess insert of 2-3 fold
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<br><br>
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<b> Note - </b>  Use 5 x more insert if  <200bps.
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<br><br>
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|-
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</td>
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| nil
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</tr>
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| nil
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</table>
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| nil
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| 2µl Deino_D
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| 2µl Deino_D
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|  2µl Agro_D
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|  2µl Agro_D
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|  2µl Agro_D
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-
 
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-
 
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|-
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| nil
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| nil
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| nil
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| 2µl Deino_E
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| 2µl Deino_E
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| 2µl Agro_E
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| 2µl Agro_E
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| 2µl Agro_E
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|-
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! scope="row"| 2. Addition of 0.05 pmol of vector
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| 2.7µl PSB-1C3
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| 2.9µl PSB-1K3
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| 2.8µl PSB-1A3
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| 2.9 µl PSB-1K3
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| 2.8 µl PSB-1A3
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| 2.7µl PSB-1C3
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| 2.9µl PSB-1K3
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| 2.8µl PSB-1A3
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|-
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! scope="row"| 3. Addition of Gibson Master Mix (µl)
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| 10
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| 10
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| 10
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| 12.9
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| 12.8
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| 12.7
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| 12.9
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| 12.8
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|-
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! scope="row"| 4. Addition of deionised H2O
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| 3.3µl
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| 3.1µl
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| 3.2µl
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| nil
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| nil
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| nil
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| nil
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| nil
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|}
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<br/>
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'''5. After addition of all components incubation at 50°C for 60 min followed. '''
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<br/>
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[[File:IMG 6181.JPG|325 px|thumb|center|Performing Gibson Assembly]]
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[[File:IMG Plan.JPG|600px|thumb|center|Planning lab work for the day]]
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[[File:IMG 6172.JPG|400 px|thumb|center|Calculations]]
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[[File:IMG 6152.JPG|400 px|thumb|center]]
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<br/>
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<br>
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<b>After addition of all components (shown in table) incubation was preformed in a thermocycler at 50°C for 60 min.</b>

Latest revision as of 10:15, 27 September 2013


Gibson Assembly Protocol


Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on NEB guidelines.


NOTE: For efficiency with reference to the NEB Gibson protocol, we considered:

1) 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
2) 0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.

A calculation provided was used furthermore to determine the pmols required based on the length of fragments:
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)

Legend

* = 50ng of 5000 bp dsDNA is approx 0.015 pmols.

** = Control reagents

*** = Additional master mix may be required for larger bp fragments.

Note - 50ng of 500 bp is approx 0.15 pmol

Note - 50-100ng of vector recommended with excess insert of 2-3 fold

Note - Use 5 x more insert if <200bps.


After addition of all components (shown in table) incubation was preformed in a thermocycler at 50°C for 60 min.

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