Team:SCUT/Protocol
Project
Related experimental method of molecular construction
1. Acquisition of the purpose gene
1.1 Objective gene extraction and PCR amplification
Use the DNA Mini Kit to extract the whole genome of the purpose strain. Please refer to the instruction of the DNA Mini Kit for the details of the procedure.
Precede PCR using the whole genome as the DNA template. The reaction system is as follows:
Table 1
The parameters of reaction condition:
Table 2
Use agarose gel electrophoresis to separate and analyze the PCR product. And then, use DNA Gel Extraction Kit to extract the purpose DNA fragment.
1.2 DNA agarose gel electrophoresis:
1.2.1 Casting a gel
1)Weigh 0.25g agarose in a conical flask. Add 1×TAE 25mL, cover a aluminized paper and dissolve the agarose in a microwave oven for 1~2min taking care not to boil the solution out of the flask.
2)Swirl to mix the gel.
3)Install the gel tray and the comb exactly.
4)Let the agarose solution cool down. Once the solution is touchable, add the DNA stain. Check the stock concentration and add the appropriate amount to give the desired final concentration.
5)Pour the gel solution into the gel tray. Remove any air bubbles with a pipette tip. Put in comb.
6)The gel will solidify while cooling down to room temperature. Remove the comb carefully and place the gel tray into the buffer chamber. Add 1x TBE buffer until the gel is completely covered.
1.2.2 Running the gel:
1)Take part of your DNA samples (~0.2 µg) and mix with loading dye. This can be done either in 1.5 mL tubes or, if the volumes are very small, on a piece of parafilm.
2)Load the size marker mixed in 1x loading dye (~6 µL final volume) into a well.
3)Load your samples into the other wells while writing down which lanes have which samples.
4)Put the lid onto the buffer chamber and connect it to the power supply. Make sure to put it in the right direction so that your DNA runs towards the positive (red) electrode.
5)Run the gel at 120 V for 30–60 min. Neither of the dyes should be run off the gel. If the electrophoresis runs correctly you will notice air bubbles coming from the negative (black) electrode.
6)Stop the run and bring your gel to a UV table to visualize your gel bands and take a picture of your gel. And then cut and extract the gel which contains the purpose DNA fragment. Use protective glasses. If sufficient separation was not achieved, put the gel back into the buffer chamber and run it for longer.
1.2.3 Extract the purpose DNA fragment:
Please refer to the instruction of the DNA Gel Extraction Kit for the details of the procedure, but taking care to cut the gel which just contains all purpose bands as small as possible.
2. Construct the gene circuits of expression system
Get the standard biological parts of promoter, RBS, and terminator. Assemble the plasmid from each standard biological parts and purpose gene by genetic engineering.
Digestion system: