Protocols

Preparation of plasmid DNA for Pronuclear Injections

The preparation of plasmid DNA fragments for oocyte pronuclear injections is critical to the success of your transgenic experiment. The DNA must be (1) intact; not sheared, nicked, or partially digested; 2) free of salts, organic solvents, agarose, and particulates; and (3) in the appropriate injection buffer, at the appropriate concentration.

The Transgenic/Knockout Facility will perform the final DNA purification steps; and then quantify and aliquot your injection construct.

The PI is responsible for the following steps in the purification of plasmid DNA for injection:

1. Prepare at least 10 ug (total) plasmid DNA. This should be the final amount of DNA isolated; if you anticipate a loss of 10-20% of DNA during processing, increase the starting amount accordingly.
2. Plasmid DNA should be isolated and purified by either Qiagen (Maxi or Midi prep kit, catalog #12162) or by CsCl gradient.
3. Purified plasmid DNA should be fully digested to release the injection fragment from the cloning vector. Ideally no vector DNA should be present in the final injection fragment. However, if it is not possible to remove vector sequence completely keep it to the least number of nucleotides possible.
4. Run an analytical gel of a small aliquot of the digest to demonstrate complete digestion without degradation. Deliver the DNA digest (~10 ug total plasmid DNA) and the gel picture of the completely digested DNA to the Transgenic/Knockout Facility (Howard Hall Room 581). Please clearly indicate the size of insert (injection fragment) and vector fragments on your gel picture.

The Transgenic/Knockout Facility will finish preparing your plasmid DNA for injection. This will include the following steps:

5. Isolate the injection fragment from 0.6% agarose using QiaxII (Qiagen #20051).
6. Quantify the gel-purified injection fragment against λHindIII fragments.
7. Dilute the injection fragment into microinjection buffer (MIB) to a concentration in the 1-10 ng/μL range.

Transgenic/Knockout Facility microinjection buffer (MIB) contains 10 mM Tris-HCl, pH 7.4 and 0.1 mM EDTA (sodium salt) sterilized by filtration. A low level of EDTA is used since it is toxic to embryos.

8. Filter the DNA solution using Ultra-Free spin columns (Millipore/Amicon) to remove any remaining small particulates that may clog injection pipets.
9. Aliquot the injection fragment solution into pre-washed, particulate-free tubes in 10 μL quantities suitable for a single injection session.
10. Injection DNA solution is stored at -20°C until used. DNA stored in this manner has generated transgenic founders >2 months after preparation. Any DNA fragment remaining after project completion with be destroyed by autoclaving or returned to the PI upon request.

DNA found to be toxic to oocytes upon injection will be returned to the PI and a fresh DNA preparation will be requested prior to repeat of injections.

Preparation of plasmid DNA for Electroporation
(ES Cell Transfection)

The preparation of plasmid DNA used for ES cell transfections is critical to the success of your knockout experiment. The DNA must be (1) intact; not sheared, nicked, or partially digested; 2) free of salts, organic solvents, agarose, and proteins; and (3) in an electrolyte-free solution, usually nuclease-free water.

The Transgenic/Knockout Facility will perform the final DNA preparation of your transfection construct.

The PI is responsible for the following steps in the DNA purification:

1. Prepare at least 200μg (total) plasmid DNA. This should be the final amount of DNA isolated; if you anticipate the loss of 10-20% of DNA during processing, increase the starting amount accordingly.
2. Plasmid DNA should be isolated and purified by either Qiagen (Maxi or Midi prep kit, catalog #12162) or by CsCl gradient.
3. Purified plasmid DNA should be fully digested to completely linearize the transfection plasmid preparation. Obviously, restriction sites within the targeting arms or insert regions should be avoided.
4. Run an analytical gel of a small aliquot of the digest to demonstrate complete linearization without degradation.
5. Quantify the DNA by spectrophotometry. The A260:A280 ratio should exceed 1.700 for best results upon transfection.
6. Remove the restriction enzymes(s) by phenol/chloroform extraction or Micropure-EZ enzyme remover (Millipore/Anicon catalog #42528).
7. Precipitate the enzyme-free, linearized DNA with absolute ethanol.
8. Deliver the DNA precipitate (200μg in a single tube, under absolute ethanol) and the gel picture of the completely digested DNA to the Trasngenic/Knockout Facility (Howard Hall Room 581). Provide spectrophotometric data for the linearized fragment indicating the A260:A280 ratio.

The Transgenic/Knockout Facility will finish preparing your plasmid DNA for transfection. This will include the following steps:

9. Wash the EtOH pellet with 70% EtOH (2X).
10. Resuspend the transfection DNA into nuclease-free water.
11. Aliquot the transfection DNA into prepared tubes at 2μg/μL in 25 μL (50μg) volumes. Each tube will be used for a single transfection of ~107 ES cells.
12. Transfection DNA aliquots will be stored at -20ºC until used. Any transfection DNA remaining after project completion with be destroyed by autoclaving or returned to the PI upon request.