From 2013.igem.org
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| | After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min. | | After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min. |
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| - | <thead><tr><th>Element </th><th>ChlM</th><th>ChlI1</th><th>CTH1</th><th>POR</th><th>ChlG</th><th>DVR1</th><th>ChlP</th><th>ChlI2</th><th>Gene</th><th>Gene</th></tr></thead>
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| - | <tbody><tr><td>Fragment 1</td><td>3.3uL Gblock1</td><td>5.0uL Gblock1</td><td>2.3uL Gblock1</td><td>4.8uL Gblock1</td></tr>
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| - | <tr class="alt"><td>Fragment 2</td><td>3.3uL Gblock2</td><td>3.3uL Gblock2</td><td>3.2uL Gblock2</td><td>3.0uL Gblock2</td></tr>
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| - | <tbody><tr><td>Fragment 3</td><td></td><td></td><td>3.1uL Gblock3</td><td></td></tr>
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| - | <tr class="alt"><td>10X T4 DNA ligase buffer</td><td>2 µL</td></tr>
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| - | <tbody><tr><td>Vector (0.05pmol)</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td></tr>
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| - | <tr class="alt"><td>H2O</td><td>0.7uL</td></tr>
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| - | <tbody><tr><td>Gibson Master Mix</td><td>10 uL</td><td>11 uL</td><td>11.3 uL</td><td>10.5 uL</td></tr>
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Revision as of 03:12, 27 September 2013
Gibson Assembly Protocol
Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on NEB guidlines.
NOTE: For efficiency with reference to the NEB Gibson protocol, we considered:
• 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
• 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)
The cells were appropriately transformed and concentrations were determined prior. (transformation protocol can be found with the following link)
-Transformation Protocol.
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* 50ng of 5000 bp dsDNA is approx 0.015 pmols.
- 50ng of 500 bp is approx 0.15 pmol
- 50-100ng of vector recommended with excess insert of 2-3 fold
- Use 5 x more insert if <200bps.
** Control reagents
*** Additional master mix may be required for larger bp fragments.
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After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min.
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