Team:PKU Beijing/Notebook/AND Gate 1/Input/LacI TetR 8

{{PKU_Beijing/Header}}
{{PKU_Beijing/Sidebar_Notebook}}
{{PKU_Beijing/Header2}}
[[Team:PKU_Beijing/Notebook|Notebook]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1|AND Gate 1]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1/Input|Input]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1/Input/LacI_TetR_8|Molecular cloning: strong promoter+Pcat-2M-lacI-term-lacP-GFP]]
==='''Molecular cloning: strong promoter+Pcat-2M-lacI-term-lacP-GFP'''===

Parts: J61100+K228819

'''Resource:''' <br>
Pcat-2M-lacI-term-lacP-GFP: myself, colonies, renamed as L1, L2, L3.  <br>
Promoter (J61100): from Lin Min, vector (has already digested by Pst1 and Spe1) 

==='''2009.8.8'''===

'''Plasmid mini prep:'''<br>
L1, L2, L3 

'''Double digest: '''<br>
L1, L2:<br>
{|Xba1||1uL
|Pst1||1uL
|-
|plasmid||10uL
|-
|Buffer||2uL
|-
|water||6uL
|}
37 ℃ 4 hour

==='''2009.8.9'''===

'''Gel electrophoresis:'''<br>
Products of double digest of L1, L2,  <br>
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
loading buffer and DNA dye: 6×<br>
voltage and time: 60V 5min; 120V 60min<br>
lane2: Marker <br>
lane1,3: L1, L3;<br>
[[Image:PKU_20090809_Shuke_Wu_3.JPG|400px]]<br>
The insert is about 2.4kb, and the backbone is 2.1kb.  

'''DNA Gel purification:'''<br>
The insert of L1 and L2.  

'''DNA ligation: '''<br>
{|cellpadding=5
|System||10uL
|-
|Insert||6uL
|-
|vector||2uL
|-
|water||3uL
|-
|buffer||1uL
|-
|T4 DNA ligase||1uL
|}
16℃ 4 hour<br>
Insert: L1, L2;<br>
Vertor: strong promoter, from Lin Min.  

'''Transformation: '''<br>
Products of ligation, competent cells 50uL each, <br>
Smear to LB plate with Amp 

==='''2009.8.10'''===

Every plate (P+L1/L2) is very well: more than 100 clones <br>
But many colonies are become green under the blue light, which means that the expression of LacI can not fully repressed the promoter lacP. 

==='''Result & Discussion'''===

I successfully constructed clone: Promoter-Pcat-2M-lacI-term-lacP-GFP . <br>
However, I disappointed to find that these clones are not work very well, because the GFP express significantly even on the plate (without induce)!!! <br>
The expression of lacI is still not enough to repress the lacP. 




{{PKU_Beijing/Foot}}
__NOTOC__