Protocols

DNA Preparation for Microinjection

Microinjection of your construct requires very pure DNA that is free of contaminants. DNA that is not purified properly will reduce transgenic efficiency and egg survival. Only use embryo tested and tissue culture grade reagents. Do not use expired reagent or kits.

Plasmid DNA Preparation

1.      Prepare plasmid DNA with Qiagen EndoFree Plasmid Kit (Qiagen 12362) or Qiagen Plasmid Maxi Kit (Qiagen 12162).

2.      Separate the fragment from the vector by restriction enzyme digestion. (Digest 15 – 20 ug of vector DNA)

3.      Electrophorese the sample on 0.8% agarose gel using 1x TAE buffer containing ethidium bromide 0.1 - 0.2 ug/ml.

4.      Excise the fragment under long-wave UV light using a clean scalpel.

5.      Purify the fragment:

-         For DNA fragment smaller than 10 Kb: use Qiagen Qiaquick Gel Extraction Kit (Qiagen 28704), and elute DNA in 40 ul embryo tested H2O (Sigma W1503) during the final step.

-         For DNA fragment from 10 - 40 Kb: use Qiagen QIAEX II Gel Extraction Kit (Qiagen 20021), and elute DNA in 40 ul embryo tested H2O (Sigma W1503) during the final step.

6.      Measure the DNA concentration by spectrophotometer.

7.      Check the DNA on an agarose gel by comparing an aliquot of the DNA sample with size standard of known concentration (e.g. Invitrogen High DNA Mass Ladder: Invitrogen 10496-016).

8.      Store DNA in -20oC.

9.      Submit 25 - 30 ul of DNA with concentration at least 40 ng/ul in H2O and the picture of the gel to the Transgenic Core for producing transgenic mice.

BAC DNA Preparation

Either circular BAC DNA or linearized BAC DNA can be used for making transgenic mice.

Circular BAC DNA Preparation:

We recommend using Clontech NucleoBond BAC 100 Kit (Clontech 740579 ). A detailed protocol can be found at the University of Michigan Transgenic Core website.   http://www.med.umich.edu/tamc/BACDNA.html  

Linearized BAC DNA Preparation:

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.

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 10 mM Tris pH 7.4, 0.1 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 4oC 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 stirring. 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 embryo tested water (Sigma W1503) 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 4oC and submitting the diluted sample.

Media & Solutions for BAC DNA:

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 -20oC.


ES Cell Preparation for Microinjection

1.      On injection day, feed cells with ES media 1 – 2 hours prior to trypsinization.

2.      Remove ES media and wash plate with PBS 2 times.

3.      Trypsinize cells for 5 minutes.

4.      Add 4 ml ES media and pipette cells to a single cell suspension (check under microscope).

5.      Place cell suspension in a 100 mm dish.

6.      Add 10 ml ES media and mix.

7.      Incubate cells 20 - 30 minutes at 37oC. (If you don’t use feeders, skip this step)

8.      Transfer cells to a 15 ml tube.

9.      Centrifuge cell suspension at 1,000 rpm for 5 minutes.

10.    Remove supernatant, and re-suspend cells in Blastocyst Injection Media (25 mM HEPES buffered DMEM plus 10% FBS, pH 7.4) at a concentration of 1 X 106/ml.

11.    Transfer cells to a cryovial, and place cells on ice.

12.    Please provide 5 ml Blastocyst Injection Media (25 mM HEPES buffered DMEM plus 10% FBS, pH 7.4)

13.    Deliver cells and injection media on ice to the Core between 10:30 - 11:00 am.

For non-Gladstone users, the Transgenic Core staff will meet you at the Gladstone lobby.