Team:OU Norman/Project/Notebook/ben

From 2014.igem.org

Digest for ter + Terminators (3A) 5/14/14

-All parts were pipetted into tubes in the following amounts.
ter #4:


  • 500 ng- Upstream Part (ter#4)
  • 1 μL- Eco RI
  • 1 μL- Spe I
  • 5 μL- 10x Buffer
  • 1 μL- BSA
  • 40.83 μL- PCR Water

4P #10:


  • 500 ng- Downstream Part (Terminator)
  • 1 μL- Xba I
  • 1 μL- Pst I
  • 5 μL- 10x Buffer
  • 1 μL- BSA
  • 40.33 μL- PCR Water

6D #16:


  • 500 ng- Downstream Part (Terminator)
  • 1 μL- Xba I
  • 1 μL- Pst I
  • 5 μL- 10x Buffer
  • 1 μL- BSA
  • 40.33 μL- PCR Water

psBIK3 #1:


  • 500 ng- Destination Plasmid (psBIK3)
  • 1 μL- Eco RI
  • 1 μL- Pst I
  • 5 μL- 10x Buffer
  • 1 μL- BSA
  • 39.15 μL- PCR Water

-Tubes were then placed in heat bath for 45 minutes at 42˚C.
-Enzymes were then heat killed on heat block for 15 minutes at 80˚C.

Ligation of ter + Terminators (3A) 5/14/14

-The ligations were preformed with these specifications.


  • 4 μL of upstream part
  • 4 μL of downstream part
  • 4 μL of destination backbone
  • 4 μL of 10x T4 ligase buffer
  • 1 μL of T4 ligase
  • 22 μL of PCR water

-The tubes were then incubated on the bench top for 20 minutes.
-2 sets of ligations were made for ter + 4P and ter + 6D.

Transformation of ter + Terminators (3A) 5/14/14

- 4 μL of ligation product was then added into 100 μL of competent cells for each transformation.
- Cells were then put on ice for 30 minutes.
- Cells were then heatshocked in a waterbath for 60 seconds at 42˚C.
- Cells were then placed back on ice for 5 minutes.
- 600 μL of PSI broth were then added to each tube.
- Then the tubes were incubated at 37˚C for 2 hours, at 200 rpms.
- Contents were then plated using 1:1 and 1:10 dilutions in PSI broth.


5/15/14

Libraries were made for ter + 4P and ter + 6D colonies.


5/20/14

Two lawns were made for each ter + 4P and ter + 6D.


5/22/14

AquaPlasmid DNA extraction was performed via Aquaplasmid's advised protocol.


Digest for ter + Terminators (3A) 06/03/14

-All parts were pipetted into tubes in the following amounts.
ter #4:


  • 500 ng- Upstream Part (ter#4)
  • 1 μL- Eco RI
  • 1 μL- Spe I
  • 5 μL- 10x Buffer
  • 1 μL- BSA
  • 40.83 μL- PCR Water

4P #10:


  • 500 ng- Downstream Part (Terminator)
  • 1 μL- Xba I
  • 1 μL- Pst I
  • 5 μL- 10x Buffer
  • 1 μL- BSA
  • 40.33 μL- PCR Water

6D #16:


  • 500 ng- Downstream Part (Terminator)
  • 1 μL- Xba I
  • 1 μL- Pst I
  • 5 μL- 10x Buffer
  • 1 μL- BSA
  • 39.43 μL- PCR Water

    • psBIK3 #1:


    • 500 ng- Destination Plasmid (psBIK3)
    • 1 μL- Eco RI
    • 1 μL- Pst I
    • 5 μL- 10x Buffer
    • 1 μL- BSA
    • 39.15 μL- PCR Water

    -Tubes were then placed in heat bath for 45 minutes at 42˚C.
    -Enzymes were then heat killed on heat block for 15 minutes at 80˚C.


    Ligation of ter + Terminators (3A) 06/03/14

    -The ligations were preformed with these specifications.


    • 4 μL of upstream part
    • 4 μL of downstream part
    • 4 μL of destination backbone
    • 4 μL of 10x T4 ligase buffer
    • 1 μL of T4 ligase
    • 22 μL of PCR water

    -The tubes were then incubated on the bench top for 20 minutes.
    -2 sets of ligations were made for ter + 4P and ter + 6D.


    Transformation of ter + Terminators (3A) 6/3/14

    - 4 μL of ligation product was then added into 100 μL of competent cells for each transformation.
    - Cells were then put on ice for 30 minutes.
    - Cells were then heatshocked in a waterbath for 60 seconds at 42˚C.
    - Cells were then placed back on ice for 5 minutes.
    - 600 μL of PSI broth were then added to each tube.
    - Then the tubes were incubated at 37˚C for 2 hours, at 200 rpms.
    - Contents were then plated using 1:1 and 1:10 dilutions in PSI broth.


    Digest for Ter + Terminators (3A) 06/09/14

    -All parts were pipetted into tubes in the following amounts.
    ter #4:


    • 500 ng- Upstream Part (ter#4)
    • 1 μL- Eco RI
    • 1 μL- Spe I
    • 5 μL- 10x Buffer
    • 1 μL- BSA
    • 41.42 μL- PCR Water

    4P #10:


    • 500 ng- Downstream Part (Terminator)
    • 1 μL- Xba I
    • 1 μL- Pst I
    • 5 μL- 10x Buffer
    • 1 μL- BSA
    • 40.33 μL- PCR Water

    6D #16:


    • 500 ng- Downstream Part (Terminator)
    • 1 μL- Xba I
    • 1 μL- Pst I
    • 5 μL- 10x Buffer
    • 1 μL- BSA
    • 39.43 μL- PCR Water

    psBIK3 #1:


    • 500 ng- Destination Plasmid (psBIK3)
    • 1 μL- Eco RI
    • 1 μL- Pst I
    • 5 μL- 10x Buffer
    • 1 μL- BSA
    • 39.15 μL- PCR Water

    -Tubes were then placed in heat bath for 45 minutes at 42˚C.
    -Enzymes were then heat killed on heat block for 15 minutes at 80˚C.


    Colony PCR 07/15/14

    One centrifuge tube was loaded with:


    • Master Mix- 450 μL
    • Forward Primer- 2 μL
    • Reverse Primer- 2 μL
    • PCR Water- 446 μL

    -This mix was then separated into 30 μL aliquots in PCR tubes.
    - A toothpick was then dipped into each colony and placed into each respective PCR tube.
    - This was done twice, once for ter + 6D, and once for ter + 4P.
    - PCR went for 94˚C for 5 hours and 45 minutes, 52˚C for 45 minutes, 72˚C for 9 minutes, and then incubated at 4˚C until extraction.


    Digest of ter 07/16/14

    -All parts were pipetted into tubes in the following amounts:


    • 500 ng- Upstream Part (ter#4)
    • 1 μL- Eco RI
    • 1 μL- Spe I
    • 5 μL- 10x Buffer
    • 1 μL- BSA
    • 40.83 μL- PCR Water

    -The tube was then placed in heat bath for 45 minutes at 42˚C.
    -Enzymes were then heat killed on heat block for 15 minutes at 80˚C.


    Gel of ter 07/16/14

    A gel was then run to confirm the presence of ter.

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