RRC-TPS: Preparing DNA for Electroporation: Protocol

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Preparing DNA for Electroporation

1. Purify plasmid from bacteria. We recommend the Qiagen EndoFree Plasmid Maxi kit (No. 12362) for the purification of the targeting vector plasmid from bacteria. Please follow the directions in the kit.

2. Linearize 150 ug (for single-drug selection) or 300 ug (for double-drug selection) of plasmid DNA with the appropriate restriction enzyme. Run a small fraction of the DNA on a mini-gel, along-side an uncut sample, to confirm that digestion is complete.

3. Add TE to 500-600 ul to facilitate extraction.

4. Extract the DNA with an equal volume neutral-buffered 1:1 phenol-chloroform. Make sure you use fresh phenol for maximum DNA recovery and highest cell viability in electroporation. Very carefully remove only the aqueous phase and transfer to a fresh tube, leaving behind (and discarding) about 10% of the aqueous phase along with the interface and lower organic phase.

5. Extract with an equal volume of 24:1 chloroform-isoamyl alcohol. Again, be very careful when removing the aqueous phase, leaving behind (and discarding) a portion of the aqueous phase at the interface.

6. Extract with an equal volume of chloroform.

7. Measure the the final volume. To precipitate add NaCl to a final concentration of 0.2M, mix, then add two volumes of ethanol. Wash the DNA pellet three times in 1ml ice-cold 70% ethanol. After the third wash spin the tube briefly; use a drawn-out Pasteur pipet or capillary to remove the residual ethanol at the bottom of the tube. Air dry.

8. Resuspend the DNA in sterile TE (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA) at 1 mg/ml (it can be more concentrated than this but do not make it any more dilute). Accurately measure the concentration and determine the A260/280. Bring the DNA, the mini-gel (step 2), and the spectrophotometer results to the facility.

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