User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/05: Difference between revisions

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** '''Three antibiotic Assembly'''
** '''Three antibiotic Assembly'''
# Description
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* As the previous ligations and transformations failed, we decided to change the technique from a simple ligation to a [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly| Three Antibiotic Assembly]. As 3 parts are requires:
1. Description
<br/ >
<br/ >
* As the previous ligations and transformations failed, we decided to change the technique from a simple ligation to a [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly| Three Antibiotic Assembly]. As 3 parts are required:
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-Plasmid [http://partsregistry.org/Part:pSB1C3 pSB1C3] represented in the image as the red construction and restricted with ECO RI and PST I.<br/ >
-Plasmid [http://partsregistry.org/Part:pSB1C3 pSB1C3] represented in the image as the red construction and restricted with ECO RI and PST I.<br/ >
-The luciferase represented as part 1 and restricted with ECO RI and SPE I. <br/ >
-The luciferase represented as part 1 and restricted with ECO RI and SPE I. <br/ >
-The double terminator represented as part 2 and done both from plasmid (with XBA I and PST I and also with ECO RI and XBA I) and from PCR (with XBA I and PST I). <br/ >
-The double terminator (TT) represented as part 2 and done both from plasmid (with XBA I and PST I and also with ECO RI and XBA I) and from PCR (with XBA I and PST I). <br/ >
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2. Restrictions:
# Restrictions:
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* The restriction (30μL) were done as follows:
* The restriction (30μL) were done as follows:
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-H<sub>2</sub>O ------------> 13μl<br/ >
-H<sub>2</sub>O ------------> 13μl<br/ >
 
-Buffer 2 -------> 4μl<br/ >
-Buffer 2 ------> 4μl<br/ >
 
-BSA ------------> 1μl<br/ >
-BSA ------------> 1μl<br/ >
-DNA ------------> 10μl<br/ >
-DNA ------------> 10μl<br/ >
 
-Enzyme I ------> 1μl<br/ >
-Enzyme I ----> 1μl<br/ >
-Enzyme II -----> 1μl<br/ >
 
-Enzyme II ---> 1μl<br/ >
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# Ligations
3. Ligations
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* The ligations were done for a total of 20μL, using different restriction enzymes and either the luciferase plasmid or the purified luciferase PCR product, and a double terminator (TT) amplified by PCR of the plasmid with the TT.  
* The ligations were done for a total of 20μL, using different restriction enzymes and either the luciferase plasmid or the purified luciferase PCR product, and a double terminator (TT) amplified by PCR of the plasmid with the TT.  
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* 1: luciferase plasmid + TT PCR  
1: luciferase plasmid + TT PCR  
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* 2: luciferase plasmid + TT plasmid
2: luciferase plasmid + TT plasmid
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* 3: luciferase PCR + TT PCR
3: luciferase PCR + TT PCR
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* 4: luciferase PCR + TT plasmid
4: luciferase PCR + TT plasmid
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* '''Reaction Mix'''
* '''Reaction Mix'''
-H<sub>2</sub>O -----------------------> 7μL<br/ >
-H<sub>2</sub>O --------------------> 7μL<br/ >
-Buffer ligase -----------> 2μL<br/ >
-Buffer ligase ----------> 2μL<br/ >
-Vector (plasmid) -----> 2μL<br/ >
-Vector (plasmid) -----> 2μL<br/ >
-Insert (luciferase) ---> 3μL<br/ >
-Insert (luciferase) ---> 3μL<br/ >
-Insert (double ter.) --> 5μL<br/ >
-Insert (double ter.) -> 5μL<br/ >
-Ligase --------------------> 1μL<br/ >
-Ligase -----------------> 1μL<br/ >
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* After the ligation, I transformed competent cells and plated them in tetracycline-LB plates.
4. Transformation
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<br/ >
* I transformed competent cells and plated them in tetracycline-LB plates. They grew well, so I replated them and extracted plasmid by using the Roche High Pure Plasmid Isolation Kit.
<br/ >
* Finally, I did a restriction for a total of 20μL using ECO RI and PST I, and I left them incubating at 37°C ON(overnight). The [[User:Mariana_Ruiz_Velasco_L./Notebook/IGEM_2010/Wet_lab_journal/2010/06/10| gel can be checked here]].


==Proving that the ligation was done correctly==
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* As a way to check that now I have my device complete (luciferase + double terminator), we synthesize a special primer containing a
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Ligation and Transformation of luciferase and double terminator


  • The first thing to do was to quantify the vector-insert relation for the ligation, I ran a 2% agarose gel for 50 min at 90V:



  • The lanes are as follow:


1. Ladder
2. Blank
3. Double terminator BBa_B0015
4. Restriction of luciferase
5. Negative control (water)
6. Positive control (plasmid pSB1C3 with luciferase but without restriction)


    • Three antibiotic Assembly


1. Description

  • As the previous ligations and transformations failed, we decided to change the technique from a simple ligation to a Three Antibiotic Assembly. As 3 parts are required:


-Plasmid pSB1C3 represented in the image as the red construction and restricted with ECO RI and PST I.
-The luciferase represented as part 1 and restricted with ECO RI and SPE I.
-The double terminator (TT) represented as part 2 and done both from plasmid (with XBA I and PST I and also with ECO RI and XBA I) and from PCR (with XBA I and PST I).




(Taken from http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly)

2. Restrictions:

  • The restriction (30μL) were done as follows:


-H2O ------------> 13μl
-Buffer 2 -------> 4μl
-BSA ------------> 1μl
-DNA ------------> 10μl
-Enzyme I ------> 1μl
-Enzyme II -----> 1μl


  • I left the restrictions incubating for 2hrs at 37°C and inactivated the enzymes at 65°C for 10 min. After this, I ran a gel at 90V for 1hr.



  • The lanes are:


1. Ladder.
2. Restriction (R.) of luciferase.
3. R. TT PCR.
4. R. TT plasmid with XBA I and PST I.
5. R. TT plasmid with ECO RI and XBA I.
6. R. plasmid pSB1C3.
7. Positive Control of the gel (luciferase PCR Product with ECO RI and SPEI).


3. Ligations

  • I did four with different combinations to optimize the probabilities of getting colonies transformed with the ligation.
  • The ligations were done for a total of 20μL, using different restriction enzymes and either the luciferase plasmid or the purified luciferase PCR product, and a double terminator (TT) amplified by PCR of the plasmid with the TT.


1: luciferase plasmid + TT PCR
2: luciferase plasmid + TT plasmid
3: luciferase PCR + TT PCR
4: luciferase PCR + TT plasmid

  • Reaction Mix

-H2O --------------------> 7μL
-Buffer ligase ----------> 2μL
-Vector (plasmid) -----> 2μL
-Insert (luciferase) ---> 3μL
-Insert (double ter.) -> 5μL
-Ligase -----------------> 1μL


4. Transformation

  • I transformed competent cells and plated them in tetracycline-LB plates. They grew well, so I replated them and extracted plasmid by using the Roche High Pure Plasmid Isolation Kit.


  • Finally, I did a restriction for a total of 20μL using ECO RI and PST I, and I left them incubating at 37°C ON(overnight). The gel can be checked here.

Proving that the ligation was done correctly


  • As a way to check that now I have my device complete (luciferase + double terminator), we synthesize a special primer containing a


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