Microinjection Buffer: 10mM Tris-HCl pH 7.4 (Sigma T2194) and 0.1mM EDTA (Sigma 03690) dissolved in high quality embryo tested water (Sigma W1503) and filtered thru a 0.2 micron filter.

1. Replicate plasmid DNA in a methylase-defective E. coli strain.

2. Recombinant plasmid DNA should be purified by CsCl or Qiagen column.

3. The insert must be separated from the vector by restriction digestion. Linear fragments with dissimilar ends have a higher chromosomal integration frequency than blunt ends. Minimize the amount of vector sequence as much as possible since it can significantly alter the expression of transgenes. Construct should be designed so the co-migrating bands of DNA are easily separated on the agarose gel.

4. Separate the insert from the vector on an agarose gel. Run in TAE buffer and use 0.5ug/ml EtBr (or SYBR Safe) for gel staining. Visualize the DNA with long wavelength UV light.

5. Excise the gel slice containing the gene fragment of interest and purify using Qiagen's Qiaex II or QIAquick gel extraction kit.

6. Ethanol precipitate the DNA. Add 1/10 volume of 3M Na Acetate, mix, then add 2-2.5 volumes of 100% EtOH. Incubate at -20∞C overnight. Spin 5 minutes in a microcentrifuge to pellet the DNA. Resuspend in Elutip buffer (low salt).

7. Pass the DNA through a DEAE elutip-D minicolumn (Schleicher and Schuell catalog #27370).

8. EtOH ppt. as in step 6 (except, no need to add salt). Wash twice in 70% EtOH at room temperature and dry. Resuspend in sterile miTE (10 mM Tris-Cl, 0.l mM EDTA, pH 7.4). Warm in a 37∞C bath for 5 minutes, mix to dissolve the pellet, then centrifuge for 10 minutes. Transfer the aqueous volume to a new tube, leaving behind a small volume of liquid in the bottom of the tube.

9. Estimate the DNA concentration using a low and/or high DNA mass ladder. Take picture of purified fragment. Run sample on NanoDrop to confirm concentration and get a 260/280 ratio.

10. Aliquot DNA, seal tube with parafilm, and store at -20∞C.

Margaret Bencsik
SF VAMC, Endocrine Research Unit
bencik@itsa.ucsf.edu

BAC Microinjection Buffer: 10 mM Tris-HCl pH 7.5 (Sigma T2194), 0.1 mM EDTA (Sigma 03690), 30 µM spermine, 70 µM spermidine, 100 mM NaCl.

1000x Polyamine Stock: 30 mM Spermine (Sigma S-1141), 70 mM Spermidine (Sigma S-2501) dissolve in embryo tested water (Sigma W1503), filter sterilize (0.2 micron filters), aliquot and store at -20 C.

1. Prepare plasmid DNA using Endofree Qiagen Maxi kit following the manufacturer's protocol. Try to minimize shear forces on the DNA.

2. Use restriction enzymes to cut out the fragment of interest, digesting about 20 µg of DNA. Slowly load the digest into a 12.5 cm trough well of a 1% Seaplaque GTG agarose (FMC), 1 X TAE gel using pipet tips that have been cut at a slant. Do not add ethidium bromide to the gel or buffer. Run the gel at 25% less voltage than you would run a TBE gel. For constructs larger than 20 Kb you may want to load your samples into a pulse field gel. See my protocol on constructs ≥ 40 Kb for details on this.

3. After running, stain the gel for 30 min. using µg/ml ethidium bromide. Do this in an opaque container covered with aluminum foil.

4. Using a hand-held long-wavelength UV light, cut out the band of interest. Trim where possible. If you decide to stop here for the day, wrap the sample in aluminum foil and store at 4∞C.

5. Digest the agarose using Gelase (Epicentre Technologies) using a 5 fold excess of enzyme. Follow the manufacturer's "High Activity Protocol".

6. Transfer the sample into 1.5 ml eppendorf tubes and microfuge at 4∞C for 5 min. to remove undigested particles of agarose. Carefully transfer the sample to 30 ml siliconized Corex tube(s). If the volume of the agarose pellet is more than 10% of your sample volume, you may be in trouble. Your sample may be prone to clogging microinjection needles. Add more Gelase to your sample and digest further if this is the case.

7. Measure your sample volume and add a half volume of room temperature 7.5 M ammonium acetate (Sigma). For every ml of sample plus ammonium acetate, add 2.5 ml of room temperature EtOH. Mix and let sit several hours or overnight at room temperature.

8. Centrifuge using a rubber adapter at 10,000 rpm and 20∞C for 30 minutes. Carefully pour off the supernatant and remove remaining liquid with cotton swabs. You should see a pellet that is probably not all DNA.

9. Air-dry the pellet until it becomes transparent. Add 100 µl of microinjection buffer (5 mM Tris pH 7.4, 0.2 mM EDTA) and gently dissolve the pellet using time and hand rocking. The last part of the pellet will probably slide off it's mooring before it is truly dissolved, so it will be necessary to very gently pipet the sample back and forth a few times using a large, slant cut pipet tip to get it into solution.

10. Pour 100 ml of microinjection buffer into 150 mm plastic petri dish. Add a micro stir bar and start staining. Float a Millipore membrane cat. #VSWP04700 on the buffer with the most shiny side up. Pipet your sample onto the center of the membrane and let it dialyze for exactly 30 minutes. Always use endotoxin tested, bottled tissue culture water to make your microinjection buffer.

11. At the end of exactly 30 minutes, pipet your sample into an eppendorf tube. It is now ready for submission for microinjection. I think it best to determine it's concentration and then dilute some of it to 2 ng/µl, keeping the main stock at 4∞C and submitting the diluted sample.

BAC Microinjection Buffer: 10 mM Tris-HCl pH 7.5 (Sigma T2194), 0.1 mM EDTA (Sigma 03690), 30 µM spermine, 70 µM spermidine, 100 mM NaCl.

1000x Polyamine Stock: 30 mM Spermine (Sigma S-1141), 70 mM Spermidine (Sigma S-2501) dissolve in embryo tested water (Sigma W1503), filter sterilize (0.2 micron filters), aliquot and store at -20 C.

1. Prepare plasmid DNA using Endofree Qiagen Maxi kit following the manufacturer's protocol. Make every effort to minimize, shear forces on the DNA.

2. Use restriction enzymes to cut out the fragment of interest, digesting about 20 µg of DNA. Slowly load the sample into a preparative low-melt pulse field gel using micropipet tips cut at a slant. Use 1% Seaplaque GTG agarose (FMC) or other high quality low-melt agarose. Run using 0.5X TBE buffer. Never use ethidium bromide in the running buffer or gel when running a pulse field gel.

3. When the gel is done running, cut out two strips of the gel about 0.5 cm wide lengthwise from the gel. The strips should be about 3 cm in from both ends of the sample trough.

4. Stain these slices 30 minutes in 1 µg/ml ethidium bromide. Visualize the DNA using long wave UV light and make a notch in the slices where the DNA band is.

5. Reassemble the gel with the slices carefully inserted exactly where they came from in the gel. Cut out the unstained DNA using the notches as your guide. Discard everything except the agarose which hopefully contains your unstained DNA. If you were directed to this protocol from the >10 Kb & <40 Kb protocol, you should now return to step 6 of that protocol.

6. Digest the agarose using Gelase (Epicentre Technologies) using a 5- 10 fold excess of enzyme for 2-3 hours. Follow the manufacturer's "High Activity Protocol".

7. Add 2 ml of endotoxin-free water to a Centricon 50 concentrator (Amicon) and centrifuge at less than 5000g (6000 rpm in an SS-34 rotor) until volume is about 100 µl. This should take about 10 minutes. Use a fixed-angle rotor such as the SS34. Use a rubber adapter and paper towels to cushion the concentrator.

8. Add 1.9 ml of your sample to the concentrator and centrifuge again until only 100 µl are left. This may take around 20 minutes. Repeat with the remainder of your sample. Now, add 1.9 ml of microinjection buffer and centrifuge as before. Microinjection buffer for constructs at least 40 Kb in size is 5 mM Tris pH 7.4, 0.2 mM EDTA, 100 mM NaCl, 30 µM spermine, and 70 µM spermidine.

9. When the 100 µl volume has again been achieved, let your sample sit quietly in the concentrator at 4∞C for an hour to wriggle free from the membrane. Then, place the concentrator inverted upside down into the collector cup and centrifuge at 500 g for 2 minutes.

10. Pour 100 ml of microinjection buffer into a 150 mm plastic petri dish. Add a micro stir bar and start stining. Float a Millipore membrane cat. #VSWP04700 on the buffer with the most shiny side up. Pipet your sample onto the center of the membrane and let it dialyze for exactly 30 minutes. Always use endotoxin tested, bottled tissue culture water to make your microinjection buffer.

11. At the end of exactly 30 minutes, pipet your sample into an eppendorf tube. It is now ready for submission for microinjection. I think it best to determine it's concentration and then dilute some of it to 2 ng/µl, keeping the main stock at 4∞C and submitting the diluted sample.

Media & Solutions Protocols

Microinjection Buffer: 10mM Tris-HCl pH 7.4 (Sigma T2194) and 0.1mM EDTA (Sigma 03690) dissolved in high quality embryo tested water (Sigma W1503) and filtered thru a 0.2 micron filter.

BAC Microinjection Buffer: 10 mM Tris-HCl pH 7.5 (Sigma T2194), 0.1 mM EDTA (Sigma 03690), 30 µM spermine, 70 µM spermidine, 100 mM NaCl.

1000x Polyamine Stock: 30 mM Spermine  (Sigma S-1141), 70 mM Spermidine (Sigma S-2501) dissolve in embryo tested water (Sigma W1503), filter sterilize (0.2 micron filters), aliquot and store at -20 C.