Protocol 1: Isolation of DNA inserts for microinjection
(Provided by Jenkins (ABL)/Cleveland (JHU) Laboratories 12/94) Solutions (All solutions are 0.2 micron filter sterilized):
Low Salt Buffer
0.2 M NaCl
20 mM TRIS (pH 7.5)
1 mM EDTA
High Salt Buffer
1.0 M NaCl
20 mM TRIS (pH 7.5)
1 mM EDTA
Injection Buffer (Provided by the Transgenic Core Lab)
10 mM TRIS (pH 7.5)
0.1 mM EDTA
1. Digest 10-20 µg of DNA with restriction enzyme to release insert.2. Separate DNA on a preparative agarose gel (usually 0.8%) in 1X TAE buffer.3. Electroelute the fragment:
a. Prepare dialysis bag:
· rinse inside and out with sterile dH20 2-3X.
· rinse inside with 1X TAE +3 mg/ml BSA (nuclease-free).
· rinse inside with 1X TAE.
· store in 1X TAE 4° C for up to 1 week.
b. Cut desired DNA band from gel, trim excess agarose, place in dialysis bag (the gel slice should be parallel to the long axis of the dialysis bag) with 2.5 to 3 ml of 1X TAE.
c. Place in electrophoresis apparatus. (DNA should be closer to the anode (+) side of the bag.) Run at 150 V until DNA is against the dialysis bag (15-60 minutes). Visualize only with long wave UV light.
d. Remove bag from apparatus, carefully remove gel slice and buffer from bag and save. Elute DNA by rinsing bag 2X with 2-4 ml of LOW SALT buffer and pool all washes.
4. Pass DNA through Elutip.
a. Prepare Elutip. Pass 2-3 ml of HIGH SALT buffer through Elutip, using a 5 ml syringe and slow steady pressure. (Intense pressure will compact the column.) Pass 5 ml LOW SALT buffer through Elutip using a 10 ml syringe.
b. Apply DNA washes to column using 10 ml syringe and slow steady pressure.
c. Rinse Elutip with 5 ml LOW SALT buffer.
d. Elute DNA with HIGH SALT buffer using minimal volume (125µl - 400 µl).5. Phenol - chloroform acetate extract 1X, chloroform acetate extract 1X.
6. Ethanol precipitate DNA 1X:
a. Add 2X volumes of 100% ethanol (DO NOT ADD SALT).
b. Centrifuge 10 min at 40 (high speed); remove supernatant.
c. Wash pellet 2X with 70% room temp ethanol to remove excess salt (which is toxic to embryos).
7. Resuspend DNA in injection buffer (10 mM TRIS/HCl 0.1 mM EDTA pH 7.5). Aliquots of embryo-tested injection buffer are available from the Transgenic Facility.8. Run the DNA on an agarose gel. Verify purity and determine concentration of DNA. Submit a photograph of the gel and 2-5 µg of insert DNA at a concentration of 100 ng/µl in injection buffer to the Transgenic Facility.