Start Time: 9:03 PM

Purpose:

To cut the gene sequences that were procured from Twist so that the desired sticky ends created using PST1 and ECOR1 will flank both the insert and plasmid backbones.

Protocol:

1. We filled a .2 mL PCR tube with 15.5 μL of milliQ water, 2.5 μL of 10x TAE buffer, 5 μL of 200 ng/μL of the gene sequence coding for Reductase, 1 μL of ECOR1, and 1 μL of PST1 in that order respectively.
2. We then filled a second .2 mL PCR tube with 15.5 μL of milliQ water, 2.5 μL of 10x TAE buffer, 5 μL of 200 ng/μL of the gene sequence coding for Desaturase, 1 μL of ECOR1, and 1 μL of PST1 in that order respectively.
3. We mixed the tubes by flicking them, and then spun the tubes in a mini centrifuge for three seconds.
4. The PCR tubes were then placed in a thermocycler that ran at 37 degrees celsius for 59 minutes and then 80 degrees celsius for 20 minutes.
5. Then we filled a third .2 mL PCR tube with 10.5 μL of milliQ water, 2.5 μL of 10x TAE buffer, 10.5 μL of 1.5 ng/μL PRS416 of the gene sequence coding for Reductase, 1 μL of ECOR1, and 1 μL of PST1 in that order respectively.
6. Then we filled a fourth .2 mL PCR tube with 10.5 μL of milliQ water, 2.5 μL of 10x TAE buffer, 10.5 μL of 1.5 ng/μL PRS314 of the gene sequence coding for Reductase, 1 μL of ECOR1, and 1 μL of PST1 in that order respectively.
7. We mixed the tubes by flicking them, and then spun the tubes in a mini centrifuge for three seconds.
8. The PCR tubes were then placed in a thermocycler that ran at 37 degrees celsius for 59 minutes, then 80 degrees celsius for 19 minutes.

Results: The PCR tube containing digested Desaturase was labeled D, the PCR tube containing digested Reductase was labeled R, the digested PRS314 was labeled 14 and the digested PRS416 was labeled 16.

Notes: Thermal cycling was done in two separate thermal cyclers.

Stop Time: 11:10 PM

Next Time: Ligate the digested backbones and inserts together to create two circular plasmids. One plasmid with the Reductase gene sequence and the prs416. The second plasmid will contain the Desaturase gene sequence and utilize the prs314 backbone.

Start Time: 11:13 PM

Purpose: To create two circular plasmids each containing a constitutive promoter (TDH3), a terminator (ADH1), and their own respective gene sequences: Reductase transcription and Desaturase transcription. Vector one designed to carry the Desaturase gene sequence with the PRS314 backbone is kept in a PCR tube labeled D14, and the vector carrying the Reductase gene combined with the PSR416 backbone is labeled R16.

Protocol:

1. The following reagents were mixed in a 0.2 mL PCR tube in order for each of the respective vectors:  

Digested DNA sample 1 (PSR314 or PSR416 Backbone) 2 μL

Digested DNA sample 2 (Desaturase or Reductase Gene Sequence) 6 μL

10x T4 DNA Ligase Buffer 1 μL

T4 DNA Ligase 1 μL

2. The reaction was run in the thermal cycler using the "igem_lig" program.

3. Note was taken that the ligation product can be used immediately or stored in the iGEM freezer.

Products: The following Table describes the samples produced:

Sample Label	Source Sample Label	Description
R16	R and 16	Purified plasmid containing PSR416 backbone, Desaturase gene sequence, ADH1 terminator, and TDH3 constitutive promoter.
D14	D and 14	Purified plasmid containing PSR314 backbone, Reductase gene sequence, ADH1 terminator, and TDH3 constitutive promoter.

Results: Success or failure.

Notes: Thermal cycling was done in two separate thermal cyclers. Results will be determined in the following protocol.   

Stop Time: 12:38 AM 

Next: Some of the circular plasmid DNA in each of the two PCR tubes generated in this protocol will be removed and gel electrophoresis will be performed on each sample so that we can determine if ligation was successful.

Start Time: 12:40 am

Purpose: Determine whether ligation of the digest product(s) was successful through gel electrophoresis.

Protocol:

1. 0.5 g agarose powder was measured out and added to a 250 ml flask
2. 50 ml 1x TAE buffer was added to the flask
3. The agarose was dissolved in a microwave until the solution became clear
4. The solution was allowed to cool until the flask could be grabbed with a bare hand (50-55°C)
5. 4 µL of ethidium bromide was added to the solution and mixed by swirling. Gloves were worn since ethidium bromide is a known carcinogen.
6. The melted agarose solution was placed into the casting tray and allowed to cool until opaque (15 minutes)
7. Enough 1X TAE buffer was added so that at least 3 mm of buffer solution covered the gel and both reservoirs at each end of the apparatus.
8. 4 µL of each sample was mixed with 0.8 µL of 6x loading dye to produce the samples that were loaded into the gel. 10 µL of the 10 kb ladder was loaded alongside the samples.
9. The gel was run at 135 V for 27 minutes, until the loading dye was halfway down the gel.  

Gel Loading Diagram: (Left to Right)

Well 1	Well 2	Well 3	Well 4	Well 5	Well 6	Well 7
10 kb ladder	D	14	D14	R	16	R16

Results:

Gel electrophoresis results indicate potential successful ligation. However, it can be concluded that ligations did not fully occur due to the presence of additional bands, which was somewhat expected. There was more insert than vector in the reaction mixture.

Notes: N/A

Next: Perform chemical transformation of E. coli with the ligated product in D14 and R16 for Desaturase and Reductase transcription genes to rapidly reproduce these genes.