Difference between revisions of "Part:BBa K649200:Experience"
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
 
===Applications of BBa_K649200===
 
===Applications of BBa_K649200===
−
The ''in vitro'' assay of BBa_640200 allowed us to confirm that the Cre-mediated recombination on lox2272 cassette works as designed.  In the assay, the part was made linear using restriction enzymes. Cre recombinase (1,000 units/ml, 1μl) was added to the linear DNA (50ng/μl, 4 μl) and incubated for 0.5, 2, and 4 hours.  Images of the experiments have been added below.
+
In in vitro assay, the part was made linear using restriction enzymes. Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours.  Images of the experiments have been added below.<br />
 
   
 
   
−
IMAGES
+
'''[Sample]'''<br />
 +
<table border="1">
 +
<tr>
 +
  <th>sample</th>
 +
  <th>volume</th>
 +
</tr>
 +
<tr>
 +
  <td>PlacIQ-lox2272-GFP-lox2272(pSB1C3)</td>
 +
  <td>50 ng/μl × 4 μl</td>
 +
</tr>
 +
<tr>
 +
  <td>10× Cre recombination Buffer</td>
 +
  <td>1 μl</td>
 +
</tr>
 +
<tr>
 +
  <td>ddH2O</td>
 +
  <td> 4 μl</td>
 +
</tr>
 +
<tr>
 +
  <td>Cre recombinase</td>
 +
  <td>1,000 units/ml × 1 μl</td>
 +
</tr>
 +
<tr>
 +
  <th>total</th>
 +
  <th> 10 μl</th>
 +
</tr>
 +
</table>
 +
 
 +
 
 
   
 
   
−
These images of the electrophoresis experiments show that there are several bands in samples to which Cre was added, which indicates that excision of the lox sites successfully occurred.
+
<br />
 +
'''[Method]'''
 +
1. The part on pSB1C3 was made linear by EcoRV restriction site which is far from lox sites. <br />
 +
2. Three identical samples and one negative control without Cre-recombinase had been prepared and those were incubated in 37°C for 0.5hr, 2hr and 4hr respectively. Negative control was incubated for 4hr.<br />
 +
3. After a period of time, the samples were kept in -20°C until the whole samples are arranged. The result was checked by electrophoresis(0.8% EtBr(+) agarose gell, 100V, 400mA, 30min)
 +
 
 
   
 
   
 
===User Reviews===
 
===User Reviews===

Revision as of 08:41, 4 October 2011

Applications of BBa_K649200

In in vitro assay, the part was made linear using restriction enzymes. Cre recombinase was added to the linear DNA and incubated for 0.5, 2, and 4 hours. Images of the experiments have been added below.

[Sample]

sample volume
PlacIQ-lox2272-GFP-lox2272(pSB1C3) 50 ng/μl × 4 μl
10× Cre recombination Buffer 1 μl
ddH2O 4 μl
Cre recombinase 1,000 units/ml × 1 μl
total 10 μl



[Method] 1. The part on pSB1C3 was made linear by EcoRV restriction site which is far from lox sites.
2. Three identical samples and one negative control without Cre-recombinase had been prepared and those were incubated in 37°C for 0.5hr, 2hr and 4hr respectively. Negative control was incubated for 4hr.
3. After a period of time, the samples were kept in -20°C until the whole samples are arranged. The result was checked by electrophoresis(0.8% EtBr(+) agarose gell, 100V, 400mA, 30min)


User Reviews

UNIQ1ac6f832977585f4-partinfo-00000000-QINU UNIQ1ac6f832977585f4-partinfo-00000001-QINU