Team:Uppsala-Sweden/Project/Protocol

Protocol

The protocols are listed in order of implementation.

Buffers, medium and solutions

Preparing chemically competent TOP10 cells

Transforming TOP10 competent cells

Inoculation

Plasmid extraction

3A assembly

Agarplate preparation

Agarose gel electrophoresis

Running agarose gels

PCR protocols

Gibson Assembly

Preparing chemically competent TOP10 cells

Materials

• Detergent-free, sterile glassware and plastic ware (see procedure)

• Table-top OD600nm spectrophotometer

• SOB medium, SOC medium

• CCMB80 buffer

CCMB80 buffer

• 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)

• 80 mM CaCl2.2H2O (11.8 g/L)

• 20 mM MnCl2.4H2O (4.0 g/L)

• 10 mM MgCl2.6H2O (2.0 g/L)

• 10% glycerol (100 ml/L)

• If pH is above 6.4, adjust pH DOWN to 6.4 with HCl

Adjusting pH up will precipitate manganese dioxide from Mn containing solutions

• Sterile filter and store at 4°C

• Slight dark precipitate appears not to affect its function

SOB-medium

SOB Medium. Used in growing bacteria for preparing chemically competent cells.

Ingredients

• 0.5% (w/v) yeast extract

• 2% (w/v) tryptone

• 10 mM NaCl

• 2.5 mM KCl

• 20 mM MgSO4

Per liter:

• 5 g yeast extract

• 20 g tryptone

• 0.584 g NaCl

• 0.186 g KCl

• 2.4 g MgSO4

Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4.

Adjust to pH 7.5. This requires approximately 25 ml of 1M NaOH per liter. Autoclave the bottled SOB

SOC medium is SOB medium with 20 mM glucose. You can do that by adding 20 ml of 20% glucose into 1 liter of SOB. Make sure everything is sterile though.

Competent Cell Preparation Procedure

Preparing glassware and media

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 75% with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Pre-chill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

• Streak TOP10 cells on an plate and grow for single colonies at 37°C
  • Room temperature works well
  • Pick single colonies into 2 ml of LB medium and shake overnight at 37°C

• Add glycerol to 15%

• Aliquot 1 ml samples to Nunc cryotubes, just Eppendorf tubes.

• Place in -80°C freezer indefinitely.

Preparing competent cells

• Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 37°C to an OD600nm of 0.3 – 0.7
  • This takes approximately 2 – 4 hours.
  • Controlling the temperature makes this a more reproducible process, but is not essential.
  • Room temperature will work. You can adjust the temperature to fit your schedule
  • Aim for lower, not higher OD if you can't hit this mark (do not miss exponential phase!)

• Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
  • Flat bottom centrifuge tubes make the fragile cells much easier to resuspend, but Falcon tubes work just fine.

• Gently resuspend in 80 ml of ice cold CCMB80 buffer
  • Sometimes, this is less than completely gentle. It still works.
  • It is often easier to resuspend pellets by stirring into 1-2 ml of buffer first, before adding large amounts of buffer to the desired final volume

• Incubate on ice 20 minutes
• Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.

• Test OD of a mixture of 950 μl SOC (Follow directions to make 1 liter of SOB media and add 50 μl 20 ml filter sterilized 20% glucose solution to the resuspended cells).
  • SOB should work just fine, SOC is just SOB with some glucose

• Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
  • If you had 2 ml of resuspension, add 1 ml more CCMB80 will be just about right

• Incubate on ice for 20 minutes
• Aliquot 75 μl to chilled Eppendorf tubes

• Store at -80°C indefinitely.
  • Flash freezing does not appear to be necessary

• Test competence (see below)

• Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x in total after several freeze/thaw cycles.

Measurement of competence (only measurement)

• Thaw the cells on ice for 15 minutes

• Transform 75 μl of cells with 2 μl of standard pUC19 plasmid (New England Biolab)
  • This was at 50 pg/μl or 10-5 μg/μl or 50 μl/ml

• Hold on ice 30 minutes
• Heat shock 60 sec at 42 °C
• Incubate on ice 5 minutes
• Add 500 μl SOC

• Incubate at 37 °C for 2 hour in 2 ml centrifuge tubes rotated
  • Using 2 ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
  • For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
  • Ampicillin and kanamycin appear to do fine with 1 hour growth

• Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
  • Good cells should yield around 100 - 400 colonies
  • Transformation efficiency is calculated as transformants / µgDNA, this variable can be easily calculated using programs online, e.g. http://www.sciencegateway.org/tools/transform.htm
  • The expected transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA

Transforming TOP10 competent cells

• Thaw the competent cells on ice for 15 minutes

• Add 1-4 μl of plasmid DNA to the 75 μl of cells.

• Incubate on ice for 30 minutes

• Heat shock 60 sec at 42 °C

• Incubate on ice 5 minutes

• Add 500 μl SOC

• Incubate at 37 °C for 2 hour in 2 ml tubes or 1.5 ml eppendorf tubes, with shaking

• Plate 200 μl, and 100 μl of 10x diluted bacteria sample on antibiotic plates using sterile 3.5 mm glass beads or glass bar

Agarplate preparation

LA (LB-Agar) – plates

15g agar into 1000 ml LB in total

• Procedure: LA -solution

First 15 g agar to 1 L flask, then add 700 ml LB Take another 1 flask and add 300 ml LB into it Autoclave both flasks 20 minutes

• When mixing LA-solution in a flask, DO NOT fill the flask with more than 75% of the flask’s max volume. Or it will boil over in the autoclave.

• If missing 1M CaCl2: Mix 29,4 g CaCl2 in 200 mL ddH20. Autoclave for 20 min.

If missing 20% glucose solution: Mix 60 g glucose to 300 ml ddH20 with a magnetic stirrer. Autoclave for 15 min.

• Take out the LA-solution from the autoclaver.

• Place the LA-solution in the 55 ºC incubators until reaching that temperature.

• When the solutions reach 55 ºC or lower stirrer the LA-soluation with a magnetic stirrer, in order to make it more homogenize.

• Add 10 ml 20% glucose and 3 ml 1M CaCl2 (DO NOT add this IF using tetracycline) to the solution in a sterile bench, preferably.

• Add antibiotics and the 300 ml autoclaved LB solution to the 700 ml autoclaved LA solution solution to obtain a total volume of 1 L.

• Mix the whole solution to make it homogenize it.

Now you can pour your plates your LABELED plates.

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.