August/11 August 2009

From 2009.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 3: Line 3:
  (plate number)-(location on plate) (no. of colonies)
  (plate number)-(location on plate) (no. of colonies)
-
               1-23L                    100>
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L]                     100>
-
               1-15N                      10
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N]                     10
-
               2-6P                      10<
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P]                       10<
-
               1-6I                      10<
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I]                       10<
-
               1-12H                      10
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H]                     10
-
               1-18C                      10<
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C]                     10<
-
               1-20F                      ×
+
               [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F]                       ×
   
   
  inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
  inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
Line 17: Line 17:
digestion with restriction enzyme
digestion with restriction enzyme
-
Vector1
+
 
-
1-2M  12
+
'''K204001'''
-
SpeⅠ 1
+
<table border="1" Frame="box">
-
PstⅠ 1
+
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
-
No.2 Buffer 2
+
<tr><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M)</td><td>12</td><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M)</td><td>5</td></tr>
-
dH2O 4
+
<tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr>
-
total 20uL
+
<tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr>
-
+
<tr><td> No.2 Buffer</td><td>2</td><td>No.2</td><td>2</td></tr>
-
Insert1
+
<tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>11</td></tr>
-
2-8M   5
+
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
-
XbaⅠ  1
+
</table>
-
PstⅠ  1
+
 
-
No.2   2
+
 
-
dH2O  11
+
-
total  20 uL
+
-
+
-
+
-
Vector2
+
-
1-23L  12
+
-
EcoRⅠ 1
+
-
XbaⅠ  1
+
-
No.2   2
+
-
dH2O  4
+
-
total 20uL
+
   
   
-
Insert2
+
'''K204002'''
-
3-18O 5
+
 
-
EcoⅠ  1
+
<table border="1" Frame="box">
-
SpeⅠ  1
+
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
-
No.2   2
+
<tr><td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)</td><td>12</td><td>[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)</td><td>5</td></tr>
-
dH2O  11
+
<tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr>
-
total 20uL
+
<tr><td>XbaI</td><td>1</td><td>SpeI</td><td>1</td><tr>
-
+
<tr><td> No.2</td><td>2</td><td>No.2</td><td>2</td></tr>
-
+
<tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>11</td></tr>
-
37℃ , 2hr
+
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
 +
</table>
 +
 
 +
<br>
 +
37°C , 2hr<br>
↓<br>
↓<br>
gel electrophoresis<br>
gel electrophoresis<br>
 +
[[Image:Osaka090811.jpg]]<br>
 +
No.1 No.2 ladder
gel cut<br>
gel cut<br>
↓<br>
↓<br>
Line 66: Line 61:
  total      50uL
  total      50uL
  ↓
  ↓
-
  16℃ overnight
+
  16°C,overnight
Line 72: Line 67:
to get new plasmid
to get new plasmid
  each 4uL(DNA) , 1-14F : 2uL
  each 4uL(DNA) , 1-14F : 2uL
-
  1-14H kan
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan
-
  1-14F kan
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F] kan
-
  2-18F Amp ('8/10competent cell
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K118013 2-18F] Amp ('8/10competent cell
   
   
-
  1-23J Amp
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp
-
  1-12C Amp (super competent cell??
+
  [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0071 1-12C] Amp (super competent cell??
 +
 
 +
 
 +
[https://2009.igem.org/Team:Osaka/NOTES back to NOTES]

Latest revision as of 07:46, 12 October 2009

1.before Min prep
Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies.

(plate number)-(location on plate) (no. of colonies)
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L]                     100>
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N]                      10
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P]                       10<
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I]                       10<
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H]                      10
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C]                      10<
             [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F]                       ×

inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)


2.digestion and ligation

digestion with restriction enzyme

K204001

VectorInsert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 RBS] (1-2M)12[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 LasR] (2-8M)5
SpeI1XbaI1
PstI1PstI1
No.2 Buffer2No.22
dH2O4dH2O11
total20uLtotal20uL


K204002

VectorInsert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)12[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)5
EcoRI1EcoRI1
XbaI1SpeI1
No.22No.22
dH2O4dH2O11
total20uLtotal20uL


37°C , 2hr


gel electrophoresis
Osaka090811.jpg

No.1 No.2 ladder

gel cut

purification by [QIAquick Nucleotide Removal Kit]

ligation

ligation
DNA        44
10* buffer 5
ligation   1
total      50uL
↓
16°C,overnight


3.Transformation
to get new plasmid

each 4uL(DNA) , 1-14F : 2uL
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F] kan
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K118013 2-18F] Amp ('8/10competent cell

[http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0071 1-12C] Amp (super competent cell??


back to NOTES