DNA- RELATED PROTOCOLS

Extraction of genomic DNA (gDNA) from D. discoideum From liquid culture:

 Put 1^.10⁶ cells in an Eppendorf tube

 Centrifuge 2 min, 4000 rpm, 21°C

 Aspirate the supernatant.

 Resuspend the cell pellet in 200 µl of SWL+ Proteinase K, and transfer in a 200 µl PCR tube.

 Lysis is made in the PCR machine, with the following program : Step 1 : 4°C 2 minutes

Step 2 : 95°C 5 minutes Step 3 : 4°C Pause

MATERIALS AND METHODS

 For a 20 µl PCR reaction, use 2 µl of this lysate.

Clones growing in 96 well plates:

 For each clone to be tested, put 10 µl of SWL+PK per PCR tube.

 Remove almost all liquid from the 96 well, but leave about 50 µl of medium (1 or 2 drops).

 Resuspend cells in this little volume and transfer 10 µl of cell suspension in the PCR tube containing SWL+PK.

 Lysis is made in the PCR machine, same conditions as above.

 For a 20 µl PCR reaction, use 2 µl of this lysate.

From colonies growing on bacteria:

 For each prep, transfer 20 µl of SWL+PK per PCR tube.

 Pick dicty from colony with a yellow tip or a toothpick and transfer in the PCR tube.

 Lysis is made in the PCR machine, same conditions as above.

 For a 20 µl PCR reaction, use 2 µl of this lysate.

PCR-GoTaq (Promega)

For KO and mutants screening: 20 µl of PCR reaction

 5x GoTaq buffer

About 1 μg of DNA is used. In the case of diagnostic digestions to screen minipreps, 5 μl of miniprepped DNA are used.

Incubate for 2 hours at 37°C. Up to 2 different enzymes were used per reaction.

MATERIALS AND METHODS

Isopropanol precipitation PCR product:

 NaOAc 0.3 M final

 0.7 volumes isopropanol

 Centrifuge max speed at 4°C for 30 min.

 Remove supernatant carefully and wash 2 times in 75% EtOH

 Let the pellet air-dry and resuspend in H2O (approx. 20 μl). Measure the concentration with Nanodrop.

Plasmid:

This protocol yield plasmid which is fine for enzymatic digestion and sequencing if the bacteria in which the plasmid is propagated are free of nucleases (TOP10, SURE, DH5a...). It will not work for bacteria still containing nucleases (MC1061P3, BL21...)

 Grow 3 ml of bacteria overnight in LB + antibiotics

 Centr. 1 ml of culture, 6000 rpm, Eppendorf centrifuge, 2 min, RT

 Aspirate supernatant with a different tip for each culture

 Vortex pellet extensively (minimum 20-30 sec)

 Add 150µl Solution 1 (from commercial miniprep kits)

 Vortex until there are no visible aggregates

 Add 150µl Solution 2, immediately mix gently by inverting tubes several times.

 Leave 5 min at RT

 Add 150µl Solution 3. Mix by inverting and shaking the tubes.

 Centrifuge 5min 10000rpm 4°C

 Collect approx. 400µl supernatant and transfer to new tubes. As much as possible try not to transfer white precipitate

 Add 800µl EtOH

 Leave minimum 5min at RT

 Centrifuge 5min, 10000rpm, 4°C

 Pour supernatant and stand tubes inverted on paper towel

 Add 1ml 70% EtOH, vortex until pellet detaches from bottom

 Centrifuge 5min, 10000rpm, 4°C

 Pour supernatant and stand tubes inverted on paper towel

 Dry the pellet. Option: if you wash with 100% EtOH drying will be very fast.

 Resuspend in 50µl TE or autoclaved water.

 For analysis we usually use 5µl for enzymatic digestion in 20µl total volume (5µl miniprep DNA, 1-2µl of enzymes, 1-2µl of 10x buffer, complete to 20µl with autoclaved water-1h 37°C)

Gel purification kit

Protocol GeneJet Gel extraction kit (Qiagen):

 Excise gel slice containing the DNA fragment using a clean scalpel or razor blade.

 Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.

Note.

 Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 µL of Binding Buffer for every 100 mg of agarose gel).

MATERIALS AND METHODS

 Incubate the gel mixture at 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process.

 Check the color of the solution. A yellow color indicates an optimal pH for DNA binding

 Transfer up to 800 µL of the solubilized gel solution (from step 3 or 4) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

 Add 700 µL of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

 Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.

 Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube (not included).

 Add 50 µL of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.

 Discard the GeneJET purification column and store the purified DNA at -20 °C.

DNA ligation New England Biolabs protocol:

 Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last

COMPONENT 20 μl REACTION

T4 DNA Ligase Buffer (10X)* 2 μl

Vector DNA (4 kb) 50 ng (0.020 pmol)

Insert DNA (1 kb) 37.5 ng (0.060 pmol)

Nuclease-free water to 20 μl

 Gently mix the reaction by pipetting up and down and microfuge briefly

 For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes

 For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation)

 Heat inactivate at 65°C for 10 minutes

 Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells Transformation competent E. coli

Preparation of cells:

 Inoculate 1l of Super-broth with 1/100 volume of a fresh O.N. culture

 Grow cells at 37°C with vigorous shaking to an OD600 of 0.5 to 0.6. It might take less than 2 hrs.

 Pour into prechilled 500 ml centrifuge flasks, cool on ice 15-30 min. Centrifuge in a cold rotor at 4000 rpm for 15 min

 Resuspend pellets in 1 liter precooled Electroporation buffer (EB)- Centrifuge as in 3-/ Repeat with 500 EB

 Resuspend in 100 ml of precooled EB- Centrifuge in the bench-top centrifuge (15 min, 3000 rpm)

 Resuspend in a final volume of 3-4 ml in precooled EB. Aliquot in precooled Eppendorf tubes, freeze in an EtOH-dry ice bath, store at -70°C. The cells are good for at least 6 months under these conditions Electrotransformation:

 Thaw the cells on ice

 In a pre-chilled eppendorf tube, mix 40 µl of cell suspension with 1 µl DNA 20ng/µl. DNA (from

MATERIALS AND METHODS

 Swith on Gene Pulser II

 Set gene pulser at 25 µF (GenePulser II Capacitance) and 1.7 kV (Gene Pulser II, press Set Volt, it will light up). Set the pulse controler to 200 Ohms on Low range, and High range to infinite (OO).

 Transfer the mixture of cells and DNA to a cold 0.1 cm electroporation cuvette and shake suspension to bottom of cuvette. Dry the cuvette very carefully and place in a safety chamber, push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.

 Pulse once by pressing the two red buttons on the Gene Pulser II until you hear a Bip (a few sec). It should produce a time constant of 4,5 to 5 ms. (The field strength will be 17 kV/cm).

 Remove the cuvette and immediately add 1 ml SOC medium (or LB) to the cuvette. (Rapid addition of SOC is important to maximize the recovery of transformants). Put back on ice.

 Transfer the cell suspension to an eppendorf tube (or a1,7x, 100mm polypropylene tube) and incubate at 37°C for 1 hour, shaking at 225 rpm.

 Centrifuge (5min 6000 rpm), aspirate medium, add 300µl SOC.

 Plate on selective medium (LB-Amp).