To transform your cells, you need three aliquots of DS941 (100ul of cells each) and you can follow the following protocol for the heat shock

To prepare the samples, you have to mix your cells with:

– aliquot 1: 10ul of your ligation reaction

– aliquot 2: 3ul of pUC19 (please ask your MSc colleagues; this is your positive control)

– aliquot 3: 10ul of sterile water (this is your negative control)

Mix the samples by gently stirring with the tip if the pipette, and let them incubate on ice for 30min, then give them a heat shock at 42°C for 45s in a heat block. It’s important this shock is immediate, so you want to keep your cells on ice the whole time, bring them close to the heated plate and transfer them directly from the ice box to the plate in a swift movement (same when you have to put them back on ice after the 45s). After this step, wait 5min, then add 400ul of LB (from one of your sterile Universal bottle) to each reaction and incubate them at 37°C in the shaker for one hour (to do so, use some tape to attach the tubes to the internal side of a plastic beaker and put the beaker in the shaker). After this time, you are ready to dilute the cells and plate them. You will need a total of 6 LB/Amp plates each and 3 LB plates without antibiotics each (you have seen where the piles are).

You have to dilute and plate:

On LB/Amp:

-DS941/ligation 100ul of a 10^-1 dilution

– DS941/ligation 100ul of cells, not diluted

– DS941/ligation all the rest of the cells (to do so, centrifuge the cells at max speed for 30s, remove the liquid with a pipette, resuspend the cells with 100ul of LB and plate them)

– DS941/pUC19 100ul of a 10^-2 dilution

– DS941/pUC19 100ul of a 10^-3 dilution

– DS941/water 100ul of undiluted cells

On LB without antibiotics (these plates will be used to count the cell):

– DS941/water 100ul of cells undiluted

– DS941/water 100ul of a 10^-3 dilution

– DS941/water 100ul of a 10^-5 dilution

Incubate all plates at 37°C until Friday, and we’ll see the results.