Team:Uppsala-Sweden/Project/Protocol

Protocol

The protocols are listed in order of implementation.

Buffers, medium and solutions

Preparing chemically competent TOP10 cells

Tranformation of TOP10

Inoculation

Plasmid extraction

3A assembly

Agarose gel electrophoresis

Running agarose gels

PCR protocols

Gibson Assembly

Agarose gel electrophoresis

Materials

• TBE buffer

• SYBR safe (10,000X stock)

• Agarose

• Microwave

Procedure

1. Add 300mL 1X TBE to a 500 mL bottle.
2. Measure out sufficient agarose to cast either a 1% (3 g) or 0.5% (1.5 g) gel.
3. Add the agarose to the TBE buffer in the 500 mL bottle.
4. Swirl to mix, do this gently since you don’t want the agarose to stick on the inner side of the bottle.
   
5. Microwave the bottle with loosened cap on high until the gel starts to bubble and is transparent. This generally takes just two minutes for 300 mL. Make sure that you stop the microwave as soon as you see bubbles. If you microwave too long, the gel will bubble over causing a big mess and you will need to start over.
6. Remove from microwave and let cool by either sitting on bench top, or use a shaker, or use a magnetic stirrer. The advantage of shaking/stirring is that even if you forget about your gel for a while, it is less likely to solidify accidentally.
7. While gel is cooling, assemble casting trays and gel combs and verify that the trays are level.
8. Once gel is cooled so the bottle can be touched comfortably with your gloved hand, add 30 μL SYBR Safe from the 10,000X concentrate stock.
9. Pour gel into casting trays. The height of the gel will depend on how much you wish to load. Diagnostic gels can be reasonably shallow since typically 10 μL volumes are loaded. For gel purifications, the gel should be much deeper.
10. Let gels sit until they are solidified.

Gels are solid when they are cloudy in appearance and firm to the touch.

Running agarose gels

Materials

• DNA ladder of the correct size range

• 1X TBE

• Loading dye

Procedure

1. Add 1X TBE buffer to gel box such that buffer just covers the top of the gel.
2. Remove comb.
3. Load 6 μL prepared ladder. Typically load ladder in left-most lane and sometimes also in right-most lane as well depending on whether you have the space.
4. Use 1 μL loading dye per 5 μL of sample.
5. Load samples left to right. The capacity of the 8 well, 1.5mm wide well is approximately 45 μL. The capacity of the 15 well, 1.5mm well is approximately 15 μL.
6. Place gel box cover on gel box such that your samples will run towards the positive, red electrode.
7. Run your gel at ~90 volts for ~60 min. The voltage and time depends on the nature of the gel as well as the samples. Use the timer to enable automatic shutoff of your gel.
8. Verify that bubbles are rising from the electrodes once you start your gel to ensure your gel is running properly.