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