Difference between revisions of "Part:BBa K2719009:Experience"

(→‎Applications of BBa_K2719009)
 
(10 intermediate revisions by 2 users not shown)
Line 7: Line 7:
 
This part was cloned on pSB1C3 using EcoRI and PstI, later it was transformed on <i>E.coli</i> DH5a (Figure 1).
 
This part was cloned on pSB1C3 using EcoRI and PstI, later it was transformed on <i>E.coli</i> DH5a (Figure 1).
  
 +
[[file:T--TecCEM--LEPCLONED.png|400px]]
 +
<p><i>Figure 1.</i> Leptin cloned in pSB1C3 in agarose gel 0.85%, 100V, 45 min.</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).
+
Then the plasmid was extracted and run on an agarose gel electrophoresis (Figure 2).   
  
−
[[file:T--TecCEM--LEPEXTRACTION.png|500px]]
+
[[file:T--TecCEM--LEPEXTRACTION.png|400px]]
 +
<p><i>Figure 2.</i> Leptin extraction in agarose gel 0.85%, 100V, 45 min.</p>
 +
 
 +
 
 +
Later an restriction was performed using EcoRV and was run on an agarose gel (Figure 3, 3.1) to probe the presence of the insert. 
 +
 
 +
[[file:T--TecCEM--LEPRESTRICTION.png|400px]]
 +
<p><i>Figure 3.</i> Leptin restriction in agarose gel 0.85%, 100V, 45 min.</p>
 +
 
 +
[[file:T--TecCEM--LEPRESTRICTIONC.png|400px]]
 +
<p><i>Figure 3.1.</i> Leptin restriction simulation (SnapGene) in agarose gel 0.85%, 100V, 45 min simulated using SnapGene.</p>
 +
 
 +
 
 +
Latter the extracted plasmid was transformed on <i>E.coli</i> BL21 (DE3).
 
   
 
   
−
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.
+
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. Then, a western blot was made for this part and positive results were obtained. (Figure 4)
  
 +
[[file:T--TecCEM--LEPMembrane.png|400px]]
 +
<p><i>Figure 4.</i> Leptin Western Blot transfer membrane results</p>
  
−
A western blot was made for this part and positive results were obtained. (Figure 5)
+
[[file:T--TecCEM--LEPWB.png|400px]]
 +
<p><i>Figure 4.1.</i> Leptin Western Blot results after a treatment with anti-histidine antibody</p>
 +
There is a band in at the expected molecular weight which suggest the production of Leptin, but it is necessary to elaborate more experiments to standardize the result.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 23:10, 17 October 2018


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_K2719009

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

T--TecCEM--LEPCLONED.png

Figure 1. Leptin cloned in pSB1C3 in agarose gel 0.85%, 100V, 45 min.

Then the plasmid was extracted and run on an agarose gel electrophoresis (Figure 2).

T--TecCEM--LEPEXTRACTION.png

Figure 2. Leptin extraction in agarose gel 0.85%, 100V, 45 min.


Later an restriction was performed using EcoRV and was run on an agarose gel (Figure 3, 3.1) to probe the presence of the insert.

T--TecCEM--LEPRESTRICTION.png

Figure 3. Leptin restriction in agarose gel 0.85%, 100V, 45 min.

T--TecCEM--LEPRESTRICTIONC.png

Figure 3.1. Leptin restriction simulation (SnapGene) in agarose gel 0.85%, 100V, 45 min simulated using SnapGene.


Latter the extracted plasmid was transformed on E.coli BL21 (DE3).

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. Then, a western blot was made for this part and positive results were obtained. (Figure 4)

T--TecCEM--LEPMembrane.png

Figure 4. Leptin Western Blot transfer membrane results

T--TecCEM--LEPWB.png

Figure 4.1. Leptin Western Blot results after a treatment with anti-histidine antibody

There is a band in at the expected molecular weight which suggest the production of Leptin, but it is necessary to elaborate more experiments to standardize the result.

User Reviews

UNIQbc453ec03593c760-partinfo-00000000-QINU UNIQbc453ec03593c760-partinfo-00000001-QINU