Team:PKU Beijing/Notebook/AND Gate 1/Output/Min Lin

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(Difference between revisions)
(New page: {{PKU_Beijing/Header}} {{PKU_Beijing/Sidebar_Notebook}} {{PKU_Beijing/Header2}} ==='''2009.9.2'''=== Enzyme digestion of the T7 promoter-9xrbs – CI –terminator with EcoRI and SpeI.<br...)
(→2009.9.2)
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==='''2009.9.2'''===
==='''2009.9.2'''===
-
Enzyme digestion of the T7 promoter-9xrbs – CI –terminator with EcoRI and SpeI.<br>
+
==='''2009.9.15'''===
-
GEL:<br>
+
-
[[Image:PKU_20090902_Min_Lin_1.JPG|400px]]
+
-
Cut GEL and purify the insert.
+
Transformation from the part distribution the P2 activator PhiR73 delta and one of its promoter PO.
-
MIniprep 1-7G, 1-7E and Bistable plasmid.<br>
+
Plate overnight.
-
Digest the 1-7G and 1-7E with EcoRI and PstI.<br>
+
-
GEL:<br>
+
-
[[Image:PKU_20090902_Min_Lin_2.JPG|400px]]
+
-
+
-
Purify the backbone.
+
-
Ligation:<br>
+
==='''2009.9.16'''===
-
9 x T7promoter-CI insert (ES)<br>
+
 
-
pSB1A2 vector(ES)
+
Pick one of the PhiR73 delta colony and PO promoter colony, shake in the incubator.
 +
 
 +
Miniprep PhiR73 delta and PO promoter plasmid.
 +
 
 +
==='''2009.9.17'''===
 +
 
 +
Enzyme Digestion of the PO promoter plasmid:
 +
{|cellpadding=5
 +
|SpeI||1ul
 +
|-
 +
|PstI||1ul
 +
|-
 +
|Buffer 3||2ul
 +
|-
 +
|Plasmid||3ul
 +
|-
 +
|ddH2O||13ul
 +
|}
 +
 
 +
 
 +
Enzyme Digestion of the E0840
 +
{|cellpadding=5
 +
|XbaI||1ul
 +
|-
 +
|PstI||1ul
 +
|-
 +
|Buffer||2ul
 +
|-
 +
|Plasmid||5ul
 +
|-
 +
|ddH2O||11ul
 +
|}
 +
 
 +
GEL assessment of the PO promoter plasmid(SP digest)
 +
 
 +
CIAP it for 20 min.
 +
 
 +
Directly purify it.
 +
 
 +
GEL: Enzyme Digestion of E0840, gel purification of the insert.
 +
 
 +
Ligation:
 +
PO promoter vector 1ul
 +
E0840 insert 7ul
 +
Ligase 1ul
 +
Ligation buffer 1ul
Transformation.
Transformation.
 +
 +
==='''2009.9.18'''===
 +
 +
Pick single colony of the PO-GFP and PCR to assess whether it is correct.
 +
All 5 of the colonies are correct.
 +
 +
Shake in the incubator.
 +
 +
Miniprep.
 +
 +
Disgest PO-GFP with XbaI and PstI, overnight.
 +
Get the Salicylate inducible promoter from zgs and digest with speI and pstI, overnight.
 +
 +
==='''2009.9.19'''===
 +
 +
GEL:
 +
PO-GFP and get the XP insert.
 +
 +
Sal promoter and pointmutation colonies for assessment.
 +
 +
Send pointmutation plasmid for sequencing.
 +
 +
CIAP the sal promoter vector for 20 min
 +
 +
Ligation:
 +
PO-GFP XP insert and sal promoter vector
 +
 +
Transformation.
 +
 +
 +
 +
==='''2009.9.20'''===
 +
 +
Pick colonies of the sal-PO-GFP
 +
 +
Colony PCR to assess. (get the correct colony, shake in the incubator)
 +
 +
Miniprep.
 +
 +
 +
 +
{{PKU_Beijing/Foot}}
{{PKU_Beijing/Foot}}
__NOTOC__
__NOTOC__

Revision as of 06:02, 16 October 2009
 

2009.9.2

2009.9.15

Transformation from the part distribution the P2 activator PhiR73 delta and one of its promoter PO.

Plate overnight.

2009.9.16

Pick one of the PhiR73 delta colony and PO promoter colony, shake in the incubator.

Miniprep PhiR73 delta and PO promoter plasmid.

2009.9.17

Enzyme Digestion of the PO promoter plasmid:

SpeI1ul
PstI1ul
Buffer 32ul
Plasmid3ul
ddH2O13ul


Enzyme Digestion of the E0840

XbaI1ul
PstI1ul
Buffer2ul
Plasmid5ul
ddH2O11ul

GEL assessment of the PO promoter plasmid(SP digest)

CIAP it for 20 min.

Directly purify it.

GEL: Enzyme Digestion of E0840, gel purification of the insert.

Ligation: PO promoter vector 1ul E0840 insert 7ul Ligase 1ul Ligation buffer 1ul

Transformation.

2009.9.18

Pick single colony of the PO-GFP and PCR to assess whether it is correct. All 5 of the colonies are correct.

Shake in the incubator.

Miniprep.

Disgest PO-GFP with XbaI and PstI, overnight. Get the Salicylate inducible promoter from zgs and digest with speI and pstI, overnight.

2009.9.19

GEL: PO-GFP and get the XP insert.

Sal promoter and pointmutation colonies for assessment.

Send pointmutation plasmid for sequencing.

CIAP the sal promoter vector for 20 min

Ligation: PO-GFP XP insert and sal promoter vector

Transformation.


2009.9.20

Pick colonies of the sal-PO-GFP

Colony PCR to assess. (get the correct colony, shake in the incubator)

Miniprep.





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