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 1: / Line 1: @@
-コロニー数カウント(8/10にトラフォ行ったもの)
+.<B>before Min prep</b><br>
- No.    コロニー数
+Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies.
--23L    100>
--15N    10
--6P     10<
--6I     10<
--12H    10
--18C    10<
--20F    ×
- 液培(5mL)にAmp 5uL , Kan 25uL入れて植菌
+ (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)
-制限酵素処理
- Vector
--2M  12
- SpeⅠ 1
- PstⅠ 1
- No.2 Buffer 2
- dH2O 4
- total 20uL
- Insert
--8M   5
- XbaⅠ  1
- PstⅠ  1
- No.2   2
- dH2O   11
- total  20 uL
+.<B>digestion and ligation</b><br>
+digestion with restriction enzyme
+'''K204001'''
+<table border="1" Frame="box">
+<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
+<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>
+<tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr>
+<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>
+<tr><td>dH<sub>2</sub>O</td><td>4</td><td>dH<sub>2</sub>O</td><td>11</td></tr>
+<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
+</table>
- Vector
+'''K204002'''
--23L  12
- EcoRⅠ 1
- XbaⅠ  1
- No.2   2
- dH2O   4
- total  20uL
- Insert
--18O  5
- EcoⅠ  1
- SpeⅠ  1
- No.2   2
- dH2O   11
- total  20uL
-時間ほど37℃で反応させた
+<table border="1" Frame="box">
+<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
+<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>
+<tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr>
+<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>
+<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
+</table>
-電気泳動
+↓<br>
-ゲル切り出し
+°C , 2hr<br>
-精製を経て
-ライゲーション
+↓<br>
-  それぞれ
+gel electrophoresis<br>
-  DNA溶液    44
+[[Image:Osaka090811.jpg]]<br>
+ No.1 No.2 ladder
+gel cut<br>
+↓<br>
+purification by [QIAquick Nucleotide Removal Kit]<br>
+↓<br>
+ligation
+  ligation
+  DNA        44
 * buffer 5
-  ライゲース 1
+  ligation   1
   total      50uL
+ ↓
+°C,overnight
-トランスフォーメーション
+.<B>Transformation</b><br>
-  各2uL , 1-14F は 4uL
+to get new plasmid
--14H kan
+  each 4uL(DNA) , 1-14F : 2uL
--14F kan
+  [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0078 1-14H] kan
--18F Amp ('8/10コンピ
+  [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
--23J Amp
+  [http://partsregistry.org/wiki/index.php?title=Part:BBa_I1466 1-23J] Amp
--12C Amp (スーパーコンピ
+  [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0071 1-12C] Amp (super competent cell??
- KanのものはLB培地を加えた後、小さな試験管に入れて振とう培養(1~2hr)してからプレートにまいた
+[https://2009.igem.org/Team:Osaka/NOTES back to NOTES]