August/11 August 2009

From 2009.igem.org

(Difference between revisions)

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

Vector		Insert
[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
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2 Buffer	2	No.2	2
dH₂O	4	dH₂O	11
total	20uL	total	20uL

K204002

Vector		Insert
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 terminator] (1-23L)	12	[http://partsregistry.org/Part:BBa_K143032 EpsE] (3-18O)	5
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	4	dH₂O	11
total	20uL	total	20uL

↓
37°C , 2hr

↓
gel electrophoresis

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

@@ Line 3: / Line 3: @@
   (plate number)-(location on plate) (no. of colonies)
--23L                     100>
+               [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 1-23L]                     100>
--15N                      10
+               [http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005 1-15N]                      10
--6P                       10<
+               [http://partsregistry.org/wiki/index.php?title=Part:BBa_K091101 2-6P]                       10<
--6I                       10<
+               [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 1-6I]                       10<
--12H                      10
+               [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0070 1-12H]                      10
--18C                      10<
+               [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 1-18C]                      10<
--20F                       ×
+               [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15009 1-20F]                       ×
   inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)
@@ Line 17: / Line 17: @@
 digestion with restriction enzyme
- Vector1
--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>
--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
--23L  12
- EcoRⅠ 1
- XbaⅠ  1
- No.2   2
- dH2O   4
- total  20uL
- Insert2
+'''K204002'''
--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>
-℃ , 2hr
+<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
+</table>
+↓<br>
+°C , 2hr<br>
 ↓<br>
 gel electrophoresis<br>
+[[Image:Osaka090811.jpg]]<br>
+ No.1 No.2 ladder
 gel cut<br>
 ↓<br>
@@ Line 66: / Line 61: @@
   total      50uL
   ↓
-℃ overnight
+°C,overnight
@@ Line 72: / Line 67: @@
 to get new plasmid
   each 4uL(DNA) , 1-14F : 2uL
--14H kan
+  [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan
--14F kan
+  [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 1-14F] kan
--18F Amp ('8/10competent cell
+  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K118013 2-18F] Amp ('8/10competent cell
--23J Amp
+  [http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp
--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]