August/10 August 2009

From 2009.igem.org

(Difference between revisions)
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3.<B>gene circuit</b><br>
3.<B>gene circuit</b><br>
digestion with restriction enzyme
digestion with restriction enzyme
 +
 +
<table width="150" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<tr>
 +
<td width="257">'''Insert 1'''</td>
 +
<td>uL</td>
 +
</tr>
 +
<tr>
 +
<td>2-16H</td>
 +
<td>6</td>
 +
</tr>
 +
<tr>
 +
<td>EcoRⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>XbaⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>10*Buffer No.2</td>
 +
<td>2</td>
 +
</tr><tr>
 +
<td>dH2O</td>
 +
<td>10</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL</td>
 +
<td>20</td></tr>
 +
</table>
 +
 +
 +
 +
<table width="150" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<tr>
 +
<td width="257">'''Vector 1'''</td>
 +
<td>uL</td>
 +
</tr>
 +
<tr>
 +
<td>1-23L</td>
 +
<td>12</td>
 +
</tr>
 +
<tr>
 +
<td>EcoRⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>SpeⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>10*Buffer No.2</td>
 +
<td>2</td>
 +
</tr><tr>
 +
<td>dH2O</td>
 +
<td>4</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL</td>
 +
<td>20</td></tr>
 +
</table>
 +
 +
 +
 +
 +
<table width="150" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<tr>
 +
<td width="257">'''Insert 2'''</td>
 +
<td>uL</td>
 +
</tr>
 +
<tr>
 +
<td>3-18O</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>EcoRⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>XbaⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>10*Buffer No.2</td>
 +
<td>2</td>
 +
</tr><tr>
 +
<td>dH2O</td>
 +
<td>11</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL</td>
 +
<td>20</td></tr>
 +
</table>
 +
 +
 +
 +
<table width="150" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 +
<tr>
 +
<td width="257">'''Vector 2'''</td>
 +
<td>uL</td>
 +
</tr>
 +
<tr>
 +
<td>1-23L</td>
 +
<td>12</td>
 +
</tr>
 +
<tr>
 +
<td>EcoRⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>SpeⅠ</td>
 +
<td>1</td>
 +
</tr><tr>
 +
<td>10*Buffer No.2</td>
 +
<td>2</td>
 +
</tr><tr>
 +
<td>dH2O</td>
 +
<td>4</td>
 +
</tr>
 +
<tr>
 +
<td>TOTAL</td>
 +
<td>20</td></tr>
 +
</table>
 +
 +
↓
 +
failured
 +
 +
 +
digestion with restriction enzyme
  insert : E+S  
  insert : E+S  
  vector : E+x  
  vector : E+x  
-
 
+
 
 +
 

Revision as of 10:07, 11 August 2009

1.Min prep
Checked the cell cultures transformed and plated out yesterday (8/9) for colony formation and made an approximate count of the number of colonies. 

(plate number)-(location on plate) (no. of colonies)
1-6O   35.2 ng/uL 
1-18L  41.8 ng/uL 
1-10K  39.1 ng/uL


2.make competent cells(with Ca)


3.gene circuit
digestion with restriction enzyme

Insert 1 uL
2-16H 6
EcoRⅠ 1
XbaⅠ 1
10*Buffer No.2 2
dH2O 10
TOTAL 20


Vector 1 uL
1-23L 12
EcoRⅠ 1
SpeⅠ 1
10*Buffer No.2 2
dH2O 4
TOTAL 20



Insert 2 uL
3-18O 5
EcoRⅠ 1
XbaⅠ 1
10*Buffer No.2 2
dH2O 11
TOTAL 20


Vector 2 uL
1-23L 12
EcoRⅠ 1
SpeⅠ 1
10*Buffer No.2 2
dH2O 4
TOTAL 20

↓ failured 


digestion with restriction enzyme
insert : E+S 
vector : E+x



4.Transformation
to get new plasmid 

1-23L A 
1-18C A 
1-20F K 
1-12H K 
1-6I  A 
2-6P  A 
1-15N A
used '8/10 competent cell
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