Difference between revisions of "Team:ASIJ TOKYO/Experiments"
| Line 212: | Line 212: | ||
<h2 class="major">Method</h2> | <h2 class="major">Method</h2> | ||
<p>Set up the following reaction on ice:</p> | <p>Set up the following reaction on ice:</p> | ||
| − | <img src="https://static.igem.org/mediawiki/ | + | <img src="https://static.igem.org/mediawiki/parts/9/94/Gibson_volumes.png" style="width:500px;height:100%;"> |
<p>* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Apply 5 times as many inserts if size falls below 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.<br/>** Control reagents are provided for 5 experiments.<br/>*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.<br/>2. Incubate samples, using a thermocycler at 50°C, for 15 minutes when 2 or 3 fragments are being assembled, or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.<br/>Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).</p> | <p>* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Apply 5 times as many inserts if size falls below 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.<br/>** Control reagents are provided for 5 experiments.<br/>*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.<br/>2. Incubate samples, using a thermocycler at 50°C, for 15 minutes when 2 or 3 fragments are being assembled, or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.<br/>Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).</p> | ||
</div> | </div> | ||
Revision as of 13:11, 31 October 2017
Competent Cell Check
The purpose of the competent cell check was to find the optimal concentration of the competent cells. We tested five different concentrations and found that 10pg/ul of the competent cells had the most efficient growth.
Materials
- Competent cell kit/ Transformation Efficiency Kit
- 2.0m; microcentrifuge
- Pipette: 1ul, 50ul, 200ul- Incubator-
Water bath- SOC media- Float rack- Agar plates
Method
1. Spin down the DNA tubes from the Competent Cell Test Kit/Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube, prior to use. A quick 20-30 second spin at 8,000-10,000 rpm will be sufficient.
Note: There should be 50 µL of DNA in each tube sent in the Kit.
2. Thaw competent cells on ice. Label one 2.0ml microcentrifuge tube for each concentration and then pre-chill by placing the tubes on ice.
3. Pipette 1 µL of DNA into each microcentrifuge tube. For each concentration, use a separate tube.
4. Pipette 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.
5. Incubate on ice for 30 minutes.
6. Preheat the water-bath to 42°C.
7. Heat-shock the cells by placing them into the water-bath for one minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
8. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.
9. Add 200 µL of SOC media per tube, and incubate at 37°C for 2 hours. Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture.
10. Pipette 20 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Do triplicates (3 each) of each tube if possible, so you can calculate an average colony yield.
11. Incubate at 37°C overnight or approximately 16 hours. Position the plates so the agar side is facing up, and the lid is facing down.
12. Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation. Make sure to measure the area of each colony to see how effective our cells are.
Gibson Assembly
The purpose of the Gibson Assembly was to synthesize DNA fragments of the E. coli bacterium with Andersen promoters of three different strengths. In performing this procedure, we were able to determine the optimal promoter strength for the growth of COX-2 and c-Myc. We discovered that the medium strength promoter was more competent with COX-2 and the strong promoter was more competent with c-Myc. The COX-2 promoter and c-Myc promoter were then synthesized by IDT and used for making the BioBrick.
Materials
- Thermocycler
- Master Mix
- Pipette: 1ul, 10ul, 11ul
- COX-2 gene
- C-Myc gene
- Anderson promoter
-
Distilled water
Method
Set up the following reaction on ice:
* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Apply 5 times as many inserts if size falls below 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
** Control reagents are provided for 5 experiments.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
2. Incubate samples, using a thermocycler at 50°C, for 15 minutes when 2 or 3 fragments are being assembled, or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
Transformation
Placeholder
Materials
Placeholder
Method
Placeholder
Electrophoresis
Placeholder
Materials
Placeholder
Method
Placeholder
Restriction Digest
Placeholder
Materials
Placeholder
Method
Placeholder
Bradford Protein Assay
Placeholder
PAGE
Placeholder
