Synthetic biology protocols

Chemocompetent bacteria:

1. Inoculate 500 ml LB with 5 ml of the overnight culture and incubate it shaking at 37 ° C till the OD = 0.4.
2. Cool the culture for 10 min. in some ice.
3. Centifruge the sample at 6000 rpm at 4 °for 3 minutes.
4. Suspend the precipitate gently in ~ 20 ml of cold solution of 0.1 M CaCl2, then add 0.1 M CaCl2 to a volume of 300 ml.
5. Centrifuge again as above.
6. Suspend the precipitate in 10 ml of cold 0.1 M CaCl2 and incubate it for 30 min.
7. Centrifuge again.
8. Suspend the sample in 6 ml of 0.1 M CaCl2 and 15% glycerol and Pipette 50 ul to an eppendorf (put them immediately in liquid nitrogen) and store at -80 °C.

Transformation of chemocompetent bacteria:

1. Put the bacteria into ice for 2-3 minutes (or until it melts)
2. Add a cooled plasmid or ligation in a volume not bigger than 20 ɰl and stir with a tip
3. Keep it in the ice for 20 to30 minutes
4. Put it to a heating block set for 42°C for 1.5 min
5. Put it back to ice for 2 min
6. Add 900 ɰl of SOB and incubate at 37°C for 1 h
7. Sow on the appropriate selective deposit.

Transformation of electrocompetent bacteria

1. Put the bacteria into some ice for 2-3 minutes (or until it melts).
2. 50 ɰl of bacteria pipete to a dialised and cooled plasmid DNA or ligation
3. Put the transformation mixture to a cuvette (also previously cooled on ice) - it is important that the sample must be on the bottom of the cuvette and free of air bubbles. You can tap the cuvette several times on the table.
4. Transform the bacteria in an electroporator set on 2500 V (time constant should be around 5)
5. Immediately add 900 ɰl of SOB and incubate at 37°C for 1 h
6. Sow the appropriate selective deposit

Cellular biology protocols

MTT assay

MTT assay

In order to measure cytotoxity of acrylamide we decided to implement MTT assay. It bases on the conversion of the tetrazolium dye- MTT to insoluable formazan by cellular enzymes. Because formazan gives a purple colour, we are enabled to measure it’s intensity at 570nm using the spectrophotometer. Cellular metabolic activity reflects the number of viable cells present in the probe so we expect a loss of purple colouring in the wells where acrylamide was added.

Protocol:

1st Day:

• Removing media

• Trypsinizing and counting cells

• Adding approximately 40 000 cells with fresh media into each well of 12-well plate

2nd Day:

• Treating cells with acrylamide: adding definite volume of stock solution of acrylamide to each well in order to obtain demanded concentration

• Adding DMSO to the control

How to find the proper amount of acrylamide?

• demanded concentration x volume (1 ml for the 12-well plate) = 10³ x sought volume

3rd Day:

• examining with pipette how much liquid remains in each well

• removing excess media, leaving only 300 µl in each well

• adding 2 times less MTT- 150 µl

• Incubating in 37°C for 2 hours

• Removing media with an aspirator

• Adding DMSO 100-500 µl transfering from each well of 12-well plate 100 µl of dissolved formazan on each well of 96-well plate. Using a few (3-5) wells of 96-well plate for each well of 12-well plate is recommended

• Reading absorbance at 570 nm

Hoechst stain

Hoechst 33342 stain protocol

In order to be able to examine cell’s nuclei morphology we introduced staining with Hoechst dye. The dye binds to the minor groove of dsDNA preferentially to sequences rich in adenine and thymine. As the Hoechst dye may be excited by ultraviolet light at around 350 nm, and then emits blue fluorescent light (maximum at around 461 nm) we’ll be able to observe changes in the cell’s nuclei using fluorescent microscopy. As UV light is harmful for the cells, they have to be fixed first in order to avoid excess death during the assay. Our aim is to study changes that occurred due to treating cells with the acrylamide: cell division, cell apoptosis and amount of micronuclei.

Protocol

Fixation of the cells:

• Remove media

• Rinse 3 times with PBS (1 ml per well of 6-well plate)

• Add 1ml of 4% paraformaldehyde and 0,5% sucrose in PBS (per well of 6-well plate

Note: Paraformaldehyde is toxic! Always use gloves, safety glasses and work under the hood while using solution.

Staining with Hoechst:

• Remove the fixative with aspirator

• Rinse 3 times with PBS

• Add 1 ml of 10.000 times diluted stock solution of Hoechst

• Incubate 30 minutes in darkness at 37°C

• Remove stain

• Rinse once with PBS then add 1 ml of PBS

Preparing a microscope preparations:

• Prepare microscopic slides

• Place a drop of mountant in the middle of the slide

• Carefully place the round glass slide on the droplet (remember to put the slide side with cells to mountant down, so the cells stay immersed)

• Remove excess liquid

Observations:

Once the microscope preparations are ready it is possible to examine them using fluorescent microscope. Hoechst dye emits blue fluorescence light at around 461 nm while excited by ultraviolet light at around 350 nm.