Team:BIOTEC Dresden/Methods Recombinase

Recombinase - Strategy

Plasmid Miniprep

Swirl the contents of the overnight culture of the bacterial cells.

Spin the cells at maximum speed for 30 seconds.

Withdraw and discard the supernatant with a pipette without disturbing the cell pellet.

Add 100µl of Buffer I (50mM Tris-HCl, 10mM EDTA, 100µg/ml RNase A, pH 8.0) to each tube and resuspend the cells by vortexing.

Add 200µl of Buffer 2 (1% SDS, 0.2M NaOH) to each tube. Close the caps and mix the solutions by inverting a few times. (do not vortex ).

Let the tubes to stand on ice for 5minutes.

Add 100µl of ice-cold Buffer 3 (3.0M Potassium Acetate, pH 5.5) to each tube. Close the tube lids and mix by inverting the tubes for a few times, with eventual formation of a white precipitate.

Let the tubes stand on ice for 5minutes and centrifuge and spin the whole solution at maximum speed for 10minutes.

Transfer the supernatant into clean 1.5ml tubes , being careful not to transfer any white precipitate.

Add about 400µl of iso-propanol to precipitate nucleic acids and let the tubes stand on ice for 10minutes.

Centrifuge the cells at maximum speed for 5minutes.

Remove and discard the supernatant .

Add 700µl of 70% ethanol to each tube and spin them at maximum speed for 2-3 minutes.

Remove and discard the supernatant carefully.

Place the tubes under fume hood for 15-20 minutes to dry off the last traces of ethanol.

Add 30µl of nuclease free water to each tube and resuspend the pellet by shaking in a shaker for 5-10 minutes.

Red/ET Recombination:

Set four eppendorf tubes with holes on the lid containing 1.4ml LB containing the same antibiotics as of the overnight culture of the concerned bacteria and inoculate with 30µl fresh overnight culture and incubate the tubes at 30˚C @950rpm for 2 hours.

Add to 2 tubes, 20µl 10% arabinose/rhamnose/anhydrotetracycline (depending on the recombination) to a final concentration of 0.1%-0.2% for inducing the expression of the recombinases. Leave the other tubes as negative controls and incubate at 37˚C @950rpm for 45mins.

Prepare electro-competent cells by putting the cells on ice, centrifuge in a cold centrifuge at 2˚C @9000rpm for 30secs, discard supernatant, add 1ml ice cold water, spin the cells @ 10,000rpm for 30secs, discard supernatant, add 1ml ice cold water, spin cells @11,000rpm for 30secs, discard supernatant keeping about 20-30µl for re-suspension.

Electroporate the plasmid of interest (pSC 101-BAD-γβαA-tet used in our experiment) by adding 1µl of the plasmid with the electro-competent cells and is electroporated @1350 volts, 10mF, 600Ohms and just after the process add 1ml LB medium to the eletroporation cuvettes and pipette back the whole solution with the cells into a fresh reaction tube and incubate at 30˚C in a shaking block for 70mins for recovery.

Plate 100µl of the recovered cells containing the antibiotic used for the Red/ET recombination and incubate the plates at 30˚C for plasmids containing pSc101 in it or at 37˚C for others.

Plating

Take blank plastic petri dishes for preparing LB plates for bacterial culture.

Add bacto agar to LB medium (1.5gms in 100ml in our experiments).

Heat the whole mixture in a microwave machine or in a autoclave still the LB heats up as much to dissolve the bacto agar in it.

After the bacto agar gets dissolved keep the whole bottle at room temperature to get cool down upto 50˚C.

After cooling add appropriate antibiotics to which the concerned bacteria is resistant in a specific concentration in the LB-agar solution.

Mix the antibiotic thoroughly with the Lb-agar.

Pour about 20ml in each blank petri dishes and allow the whole solution to solidify keeping the lid half open.

After solidification keep the plated perti dishes at 4˚C for further use.

Agarose Gel Electrophoresis

Weigh agarose in a specific amount for the desired concentration of the gel to be prepared.

Dissolve the agarose powder in a compatible buffer (Tris Buffer Acetate/Tris Buffer EDTA).

Arrange a gel apparatus with holders and a comb with adequate number of teeth for loading the sample.

Heat the mixture of agarose and buffer in a microwave or in a autoclave until all the agarose powder gets dissolved.

Cool the solution by swirling for a few times.#

Add desired concentration of ethidium bromide with the gel and mix it properly.

Pour the resultant liquid into the gel chamber and let it stand to solidify.

After solidification take out the holders and comb and pour the compatible buffer in the chamber.

Load the DNA marker on one lane and the samples in other and run the gel till the DNA gets resolved properly to be visible under UV exposure.

I have a cunning plan.