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Protocols
	DNA Purification for Microinjection   This method uses GeneClean kit to purify DNA from a low melting point agarose gel.  The kit works through the absorption of DNA onto glass beads in the presence of a chaotropic salt (NaI). The DNA can be prepared from a Qiagen maxiprep kit or from two CsCl gradients.

1)     Cut 10µg of plasmid DNA with the appropriate enzyme(s) to release the insert from the bacterial vector.

2)     Run the DNA out on a low melting point agarose gel - use GTG grade. The BRL minigel electrophoresis units work well. Load one of the small wells with appropriate amount of markers and load the digest into the remaining wells.

3)     Excise the proper band from the gel. Visualize the small gel slice on UV light box and use it to gauge where the correct band is on the larger preparative portion of the gel. Cut out the small lane of the digest without the markers.

4)     Place the gel slice in tarred Eppendorf tubes. Weigh to determine volume of gel slice. Assume 1 mg = 1µl of gel.  Keep the volume of gel slice below 400µl per tube to allow for enough room to add NaI.

5)     Add three volumes of NaI provided in the GeneClean kit. Melt at 55˚ C for 5 min.

6)     Add 5 µl of Glassmilk suspension. Invert to mix and let sit on ice to allow DNA to bind. If more than 5 µg of DNA is being purified, add 1µl of glassmilk per 0.5 µg of DNA above 5 µg. Allowing up to 30 minutes for binding will slightly increase the yield.

7)     Transfer suspension to Eppendorf tubes.  Spin down the glassmilk by microcentrifuge for 5 seconds. The capacity of the spin filter is 1.5ml, so multiple tubes may be necessary.

8)     Wash 5 to 6 times with ice cold NEW wash, emptying catch tube each time.

9)     Spin at full speed for 20 seconds to dry the glassmilk and fully remove all NEW wash.

10) Transfer the spin filter to a fresh catch tube and add 100 µl of injection buffer to elute DNA. Two 50 µl aliquots instead of 100 µl will increase yield. Heat the injection buffer (5mM Tris HCl pH 7.5, EDTA 0.1mM) to 55˚ C to increase the yield. The spin filter can be heated to 55˚ C as well.

11) Filter the DNA through a Millipore syringe filter (cat. # SJHVL04NS) with a 1 ml syringe. Quantitate the DNA by gel electrophoresis with markers before injection and dilute it to the appropriate concentration. A concentration above 25 ng/µl is optimal.

                             
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