Difference between revisions of "Part:BBa K1918104:Experience"

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===Applications of BBa_K1918104===
 
===Applications of BBa_K1918104===
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1. Use PCR to get the DNA fragment of TRE and IntC:EBFP2C(155-238)
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Firstly, we need to acquire the elements.  
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2. Ligate the fragment with vector
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We using PCR, Overlap PCR and Gibson to get the elements such as EBFP2, EBFP2N(1-154), EBFP2C(155-238), EBFP2N(1-154):IntN and IntC:EBFP2(155-238).
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3. Heat shock transformation
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4. Select colonies
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Secondly, we construct the part.
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5. Purification of plasmid DNA
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We combine the elements with the vector and transfer the vector into the competent E. coli cells. After that, we select the positive clones and use colony PCR to identify the recombinants. Culturing the positive clones and sequencing the isolated plasmids to make sure that we have get the needed parts.
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7. Sequencing
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8. Transfer into 293T cell
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Lastly, we transfect the plasmids to 289H cells and do the FACS test.
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9. Use Dox to induce the expression of IntC:EBFP2C(155-238)
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In the results of FASC test, the EBFP-A signals tell us that the split florescent proteins could fuse together and our parts work fine.
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10. FACS
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===User Reviews===
 
===User Reviews===

Latest revision as of 21:17, 19 October 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1918104

Firstly, we need to acquire the elements. We using PCR, Overlap PCR and Gibson to get the elements such as EBFP2, EBFP2N(1-154), EBFP2C(155-238), EBFP2N(1-154):IntN and IntC:EBFP2(155-238).

Secondly, we construct the part. We combine the elements with the vector and transfer the vector into the competent E. coli cells. After that, we select the positive clones and use colony PCR to identify the recombinants. Culturing the positive clones and sequencing the isolated plasmids to make sure that we have get the needed parts.

Lastly, we transfect the plasmids to 289H cells and do the FACS test. In the results of FASC test, the EBFP-A signals tell us that the split florescent proteins could fuse together and our parts work fine.

User Reviews

UNIQa1e6980e9d63e093-partinfo-00000000-QINU UNIQa1e6980e9d63e093-partinfo-00000001-QINU