Difference between revisions of "Part:BBa K649200:Experience"
 
(11 intermediate revisions by the same user not shown)
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
−
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
−
how you used this part and how it worked out.
 
−
 
 
===Applications of BBa_K649200===
 
===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.<br /><br />
−
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.
+
 
   
 
   
−
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]'''
 +
<br />
 +
1. The part on pSB1C3 was made linear by EcoRV restriction site which is far from ''lox'' sites. <br /><br />
 +
2. Three identical samples and one negative control supplied with ddH2O instead of 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 /><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<br />(0.8% EtBr(+) agarose gell, 100V, 400mA, 30min).
 +
<br />
 +
<br />
 +
[[Image:in vitro_lox_gfp_lox.png|thumb|center|500px|<br/>|''in vitro'' assay of K649200<br />negative control, 0.5hr, 2hr, and 4hr from left]]
 +
<br />
 +
These image of the electrophoresis experiment shows that there are several bands in samples to which Cre was added, which indicates that excision of the ''lox'' sites successfully occurred.
 
   
 
   
 
===User Reviews===
 
===User Reviews===

Latest revision as of 05:11, 29 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 supplied with ddH2O instead of 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).

in vitro assay of K649200
negative control, 0.5hr, 2hr, and 4hr from left


These image of the electrophoresis experiment shows that there are several bands in samples to which Cre was added, which indicates that excision of the lox sites successfully occurred.

User Reviews

UNIQ41977603b52adee0-partinfo-00000000-QINU UNIQ41977603b52adee0-partinfo-00000001-QINU