Difference between revisions of "Team:IISc-Bangalore/Assembly"

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<h1 id="pcrs">PCRs: Adding Restriction Sites and Linkers</h1>
 
<h1 id="pcrs">PCRs: Adding Restriction Sites and Linkers</h1>
 
<h1 id="restriction-digests">Restriction Digests</h1>
 
<h1 id="restriction-digests">Restriction Digests</h1>
 +
<table>
 +
<tr>
 +
<th colspan="8">Restriction enzymes used in our assembly</th>
 +
</tr>
 +
<tr>
 +
<th rowspan="2">Restriction Enzyme</th>
 +
<th rowspan="2">Sequence</th>
 +
<th colspan="4">Activity in NEBuffers (%)</th>
 +
<th rowspan="2">Incubation temperature</th>
 +
<th rowspan="2">Heat inactivation</th>
 +
</tr>
 +
<tr>
 +
<th>1.1</th>
 +
<th>2.1</th>
 +
<th>3.1</th>
 +
<th>CutSmart</th>
 +
</tr>
 +
 +
<tr>
 +
<td>AgeI</td>
 +
<td>A\CCGGT</td>
 +
<td>100</td>
 +
<td>75</td>
 +
<td>25</td>
 +
<td>75</td>
 +
<td>37°C</td>
 +
<td>65°C</td>
 +
</tr>
 +
<tr>
 +
<td>AgeI-HF</td>
 +
<td>A\CCGGT</td>
 +
<td>100</td>
 +
<td>50</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>65°C</td>
 +
</tr>
 +
<tr>
 +
<td>BamHI</td>
 +
<td>G\GATCC</td>
 +
<td>75*</td>
 +
<td>100*</td>
 +
<td>100</td>
 +
<td>100*</td>
 +
<td>37°C</td>
 +
<td>—</td>
 +
</tr>
 +
<tr>
 +
<td>BamHI-HF</td>
 +
<td>G\GATCC</td>
 +
<td>100</td>
 +
<td>50</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>—</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII</td>
 +
<td>A\AGCTT</td>
 +
<td>25</td>
 +
<td>100</td>
 +
<td>50</td>
 +
<td>50</td>
 +
<td>37°C</td>
 +
<td>80°C</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII-HF</td>
 +
<td>A\AGCTT</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>80°C</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII</td>
 +
<td>A\AGCTT</td>
 +
<td>25</td>
 +
<td>100</td>
 +
<td>50</td>
 +
<td>50</td>
 +
<td>37°C</td>
 +
<td>80°C</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII-HF</td>
 +
<td>A\AGCTT</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>80°C</td>
 +
</tr><tr>
 +
<td>NcoI</td>
 +
<td>C\CATGG</td>
 +
<td>100</td>
 +
<td>100</td>
 +
<td>100</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>80°C</td>
 +
</tr>
 +
<tr>
 +
<td>NcoI-HF</td>
 +
<td>C\CATGG</td>
 +
<td>50</td>
 +
<td>100</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>80°C</td>
 +
</tr>
 +
<tr>
 +
<td>NheI</td>
 +
<td>G\CTAGC</td>
 +
<td>100</td>
 +
<td>100</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>65°C</td>
 +
</tr>
 +
<tr>
 +
<td>NheI-HF</td>
 +
<td>G\CTAGC</td>
 +
<td>100</td>
 +
<td>25</td>
 +
<td>10</td>
 +
<td>100</td>
 +
<td>37°C</td>
 +
<td>80°C</td>
 +
</tr>
 +
<tr>
 +
<td colspan="8">* denotes star activity| NOTE ADD NdeI TOO</td>
 +
</tr>
 +
 +
</table>
 +
 
<h1 id="ligation">Multi-Ligation: Coming Full Circle</h1>
 
<h1 id="ligation">Multi-Ligation: Coming Full Circle</h1>
 
<h1 id="screening">Screening Transformants: Colony PCR with VF2/VR</h1>
 
<h1 id="screening">Screening Transformants: Colony PCR with VF2/VR</h1>

Revision as of 06:43, 31 October 2017

  1. BioBrick Transformations
  2. Plasmid Isolation
  3. PCRs
  4. Restriction digests
  5. Ligations
  6. Screening

BioBrick Transformations

Plasmid Isolation

PCRs: Adding Restriction Sites and Linkers

Restriction Digests

Restriction enzymes used in our assembly
Restriction Enzyme Sequence Activity in NEBuffers (%) Incubation temperature Heat inactivation
1.1 2.1 3.1 CutSmart
AgeI A\CCGGT 100 75 25 75 37°C 65°C
AgeI-HF A\CCGGT 100 50 10 100 37°C 65°C
BamHI G\GATCC 75* 100* 100 100* 37°C —
BamHI-HF G\GATCC 100 50 10 100 37°C —
HindIII A\AGCTT 25 100 50 50 37°C 80°C
HindIII-HF A\AGCTT 10 100 10 100 37°C 80°C
HindIII A\AGCTT 25 100 50 50 37°C 80°C
HindIII-HF A\AGCTT 10 100 10 100 37°C 80°C
NcoI C\CATGG 100 100 100 100 37°C 80°C
NcoI-HF C\CATGG 50 100 10 100 37°C 80°C
NheI G\CTAGC 100 100 10 100 37°C 65°C
NheI-HF G\CTAGC 100 25 10 100 37°C 80°C
* denotes star activity| NOTE ADD NdeI TOO

Multi-Ligation: Coming Full Circle

Screening Transformants: Colony PCR with VF2/VR