Team:OU Norman/Project/Notebook/ben

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 the Aquaplasmid 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.