Difference between revisions of "Part:BBa K2719005:Experience"

Latest revision as of 22:54, 17 October 2018

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Applications of BBa_K2719005

This part was cloned on pSB1C3 using EcoRI and PstI, later it was transformed on E.coli DH5a (Figure 1).

Figure 1. Colonies transformed with the Tenascin 5 Domain V Expression device

Then the plasmid was extracted and run on an agarose gel electrophoresis (Figure 2). Latter an restriction was performed using NotI and was run on an agarose gel (Figure 3) to probe the presence of the insert. After the extracted plasmid it was transformed on E.coli BL21 (DE3).

Figure 2. 0.85% agarose gel with GelRed: MW) 1Kb plus from NEB; 7) Tenascin 5 Domain V Expression device (BBa_K2719005) V'; 8) Tenascin 5 Domain V Expression device (BBa_K2719005) V

Figure 3. 0.85% agarose gel with GelRed: MW) 2-Log DNA Ladder; 4) Tenascin 5 Domain V Expression device (BBa_K2719005) restriction with NotI

The E.coli BL21 (DE3) colonies were later inoculated on liquid media (seed culture) and incubated overnight. From the seed culture 1 ml was inoculated on fresh LB+CAM broth, and at regular intervals the absorbance was measured. When the absorbance reached 0.7, IPTG was added to a final concentration of 1 mM and samples were taken after 5 and 16 h. Those samples were lysed. The lysate was loaded into a 18% polyacrylamide SDS-PAGE (Figure 4), from which a higher presence of protein was detected at 5 h after induction.

Figure 4. 20% polyacrylamide gel: MW) Presicion Plus Protein Standard; 7) Tenascin 5 Domain V Expression device (BBa_K2719005) protein

User Reviews

UNIQeb8e6dd94d9ddc9d-partinfo-00000000-QINU UNIQeb8e6dd94d9ddc9d-partinfo-00000001-QINU

@@ Line 10: / Line 10: @@
 <p><i>Figure 1.</i> Colonies transformed with the Tenascin 5 Domain V Expression device</p>
-<p>Then the plasmid was extracted and run on an agarose gel electrophoresis (Figure 2).  Later an restriction was performed using NotI and was run on an agarose gel (Figure 3) to probe the presence of the insert.  Later the extracted plasmid was transformed on <i>E.coli</i> BL21 (DE3).</p>
+<p>Then the plasmid was extracted and run on an agarose gel electrophoresis (Figure 2).  Latter an restriction was performed using NotI and was run on an agarose gel (Figure 3) to probe the presence of the insert.  After the extracted plasmid it was transformed on <i>E.coli</i> BL21 (DE3).</p>
+[[file:T--TecCEM--BBTCD5Extrac.png|500px]]
+<p><i>Figure 2.</i> 0.85% agarose gel with GelRed: MW) 1Kb plus from NEB; 7) Tenascin 5 Domain V Expression device (BBa_K2719005) V'; 8) Tenascin 5 Domain V Expression device (BBa_K2719005) V'' </p>
+[[file:T--TecCEM--ResK2719005.png|500px]]
+<p><i>Figure 3.</i> 0.85% agarose gel with GelRed: MW) 2-Log DNA Ladder; 4) Tenascin 5 Domain V Expression device (BBa_K2719005) restriction with NotI</p>
 <p>The <i>E.coli</i> BL21 (DE3) colonies were later inoculated on liquid media (seed culture) and incubated overnight.  From the seed culture 1 ml was inoculated on fresh LB+CAM broth, and at regular intervals the absorbance was measured.  When the absorbance reached 0.7, IPTG was added to a final concentration of 1 mM and samples were taken after 5 and 16 h.  Those samples were lysed.   The lysate was loaded into a 18% polyacrylamide SDS-PAGE (Figure 4), from which a higher presence of protein was detected at 5 h after induction.</p>
+[[file:T--TecCEM--SDSK2719005.png|500px]]
+<p><i>Figure 4.</i> 20% polyacrylamide gel: MW) Presicion Plus Protein Standard; 7) Tenascin 5 Domain V Expression device (BBa_K2719005) protein</p>
 ===User Reviews===