Difference between revisions of "Part:BBa K1429002"
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IP free cyan fluorescent protein.  We have a ribosome binding site (RBS).
 
IP free cyan fluorescent protein.  We have a ribosome binding site (RBS).
  
 +
<h4>Background</h4>
 +
<div class='entry-results'><p>We want to take Tuesday&#39;s PCR products and put them into the pSB1C3 backbone.&nbsp;</p>
 +
 +
<p><strong>Digest PCRs:</strong></p>
 +
 +
<p>10 ul PCR product</p>
 +
 +
<p>2 ul cutsmart buffer (10x stock)</p>
 +
 +
<p>1 ul PstI</p>
 +
 +
<p><u>1 ul EcoRI</u></p>
 +
 +
<p>20 ul total --&gt; incubate for 30 min at 37C</p>
 +
 +
<p>&nbsp;</p>
 +
 +
<p><strong>PCR purify digest product (only 14 ul - save 6 ul):</strong></p>
 +
 +
<p>Follow kit protocol. Elute in elution buffer.</p>
 +
 +
<p>Worried that the washed columns won&#39;t bind DNA, we are going to use some of the set-aside (unpurified) digest product for a backup ligation. We&#39;ll run a gel of our purification, but we are going to set up a ligation beforehand, so we won&#39;t have even rough estimates of DNA concentrations.</p>
 +
 +
<p>Set up ligations:</p>
  
 
<table style="width: 500px;">
 
<table style="width: 500px;">
−
+
<tbody>
 
<tr>
 
<tr>
 
<td><strong>Component</strong></td>
 
<td><strong>Component</strong></td>
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<td>&nbsp;</td>
 
<td>&nbsp;</td>
 
</tr>
 
</tr>
 +
</tbody>
 
</table>
 
</table>
  
 +
<p>The above were incubated 30 min at RT then stored at -20C.</p>
 +
 +
<p>&nbsp;</p>
 +
</div>
 +
<br>
 +
</div>
 +
<div class='entry'>
 +
<h4>Results</h4>
 +
<div class='entry-results'><p>We ran a 1% gel of the digest before and after purification. We had a decent yield, maybe 40% of our initial digest product in the purified lanes.&nbsp;</p>
 +
</div>
 +
<br>
 +
</div>
 +
<div class='entry'>
 +
<h4>Conclusions</h4>
 +
<div class='entry-results'><p>Next step: Transform the ligated plasmids into E. coli!</p>
 +
</div>
 +
<br>
 +
</div>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 01:47, 1 October 2014

Cyan Fluorescent Protein (CFP) "Cindy Lou" coding region, intellectual property-free

IP free cyan fluorescent protein. We have a ribosome binding site (RBS).

Background

We want to take Tuesday's PCR products and put them into the pSB1C3 backbone. 

Digest PCRs:

10 ul PCR product

2 ul cutsmart buffer (10x stock)

1 ul PstI

1 ul EcoRI

20 ul total --> incubate for 30 min at 37C

 

PCR purify digest product (only 14 ul - save 6 ul):

Follow kit protocol. Elute in elution buffer.

Worried that the washed columns won't bind DNA, we are going to use some of the set-aside (unpurified) digest product for a backup ligation. We'll run a gel of our purification, but we are going to set up a ligation beforehand, so we won't have even rough estimates of DNA concentrations.

Set up ligations:

<tbody> </tbody>
Component Using purified digest product Using unpurified digest product BB alone
dH2O x 11 14
Insert (RFP or GFP) 14 3 x
1:10 BB 3 3 3
T4 buffer (10 1 1 1
       

The above were incubated 30 min at RT then stored at -20C.

 


</div>

Results

We ran a 1% gel of the digest before and after purification. We had a decent yield, maybe 40% of our initial digest product in the purified lanes. 


Conclusions

Next step: Transform the ligated plasmids into E. coli!


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 74
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2