Difference between revisions of "Part:BBa K1429002"
Line 89: Line 89:
 
</div>
 
</div>
  
−
<div class='entry'>
 
−
<div class='entry-results'><p><strong>Background info:</strong></p>
 
  
−
<ul>
 
−
<li>The plasmid is about 2000 base pairs (bp). &nbsp;</li>
 
−
<li>Our PCRed GFP (gene of interest) is about 750 bp.</li>
 
−
<li>Assume that&nbsp;1 uL of this PCR purified GFP = 50 ng (info from Ellen).</li>
 
−
</ul>
 
−
 
−
<p><strong>Calculations:</strong></p>
 
−
 
−
<p>We have 6 uL of our gene of interest in a solution totaling 20 uL.</p>
 
−
 
−
<p>So we have:</p>
 
−
 
−
<p><span class="math-tex">\(\displaystyle \frac{6\mu L }{1}PCR \; purified \; GFP * \frac{50 ng}{1 \mu L} * \frac{1}{20\mu L} = \frac{300 ng}{20 \mu L} PCR \; purified \: GFP = 15\frac{ng}{\mu L}PCR \; purified \; GFP\)</span></p>
 
−
 
−
<p>&nbsp;</p>
 
−
 
−
<p>The iGEM kit provides a linearized plasmid backbone in a solution with a concentration of 25 ng/uL. &nbsp;</p>
 
−
 
−
<p>In our digestion step, we used 4 uL of the linearized plasmid backbone and 4 uL of the enzyme master mix.&nbsp;</p>
 
−
 
−
<p>So, we have:</p>
 
−
 
−
<p><span class="math-tex">\(\displaystyle \frac{4\mu L }{1}plasmid * \frac{25 ng}{1 \mu L} * \frac{1}{(4+4)\mu L} = \frac{100 ng}{8 \mu L} plasmid= 12.5\frac{ng}{\mu L}plasmid\)</span></p>
 
−
 
−
<p>We need to have a GFP:plasmid ratio of at least 3:1 to make sure that we have enough pieces of the gene of interest to successfully connect to&nbsp;the plasmid backbone.</p>
 
−
 
−
<p>Now, we&#39;ll use the <a href="http://nebiocalculator.neb.com/#!/" target="_blank">NEBioCalculator</a>&nbsp; to get the number of moles for each (see the pictures).</p>
 
−
 
−
<p>So, we have:</p>
 
−
 
−
<p><span class="math-tex">\(\displaystyle \frac{32 . 36 \frac{fmol}{\mu L} \; GFP}{9 . 632 \frac{fmol}{\mu L} \; plasmid} = 3 . 36 \; GFP : 1 \; plasmid \gt 3 \; GFP : 1 \; plasmid\)</span></p>
 
−
 
−
<p>Therefore, we can use a ratio of&nbsp;&nbsp;1 uL of the PCR purified&nbsp;GFP solution to 1 uL of the digested plasmid solution.</p>
 
−
 
−
<p>&nbsp;</p>
 
−
</div>
 
−
</div>
 
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 02:07, 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