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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<b>100% of this page is still under construction, the last big protocols page we need to finish, Ignore everything on this page for now</b>
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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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Before beginning Gibson assembly, all agar plate requirements, concentration calculations and equipment requirements were prepared. Once the gBlocks had arrived Gibson assembly was performed on all fragments.<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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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 -   
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 -   
</html>[[Team:Macquarie/Protocols/TransformationProtocol|Transformation Protocol]]. <html></p></html>
</html>[[Team:Macquarie/Protocols/TransformationProtocol|Transformation Protocol]]. <html></p></html>
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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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! scope="row" rowspan="5" width="20%" | 1. Addition of 20ng Gene Block Fragments in appropriate tube
 
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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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|  2µl Hemo_B
 
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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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| 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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|-
 
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| nil
 
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| nil
 
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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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| 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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'''5. After addition of all components incubation at 50°C for 60 min followed. '''
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'''5. After addition of all components (shown in table) incubation was preformed at 50°C for 60 min followed. '''
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Revision as of 06:33, 26 September 2013


Gibson Assembly Protocol


Before beginning Gibson assembly, all agar plate requirements, concentration calculations and equipment requirements were prepared. Once the gBlocks had arrived Gibson assembly was performed on all fragments.

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 - Transformation Protocol.




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



Element ChlMChlI1CTH1PORChlGDVR1ChlPChlI2GeneGene
Fragment 13.3uL Gblock15.0uL Gblock12.3uL Gblock14.8uL Gblock1
Fragment 23.3uL Gblock23.3uL Gblock23.2uL Gblock23.0uL Gblock2
Fragment 33.1uL Gblock3
10X T4 DNA ligase buffer2 µL
Vector (0.05pmol)2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL
H2O0.7uL
Gibson Master Mix10 uL11 uL11.3 uL10.5 uL

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