2. MATERIAL AND METHODS
2.2 Methods
2.2.1 Isolation of Nucleic Acids
2.2.1.1 Mini-Preparation of Plasmid DNA
This method was used to to produce small amounts of pure plasmid DNA. From an overnight bacterial culture 1.5 ml were transferred to an Eppendorf tube and centrifuged at 4,000x g for 10 min. The supernatant was discarded and the rest of the medium was eliminated by inversion on absorbent paper. The pellet was resuspended in 150 µl of buffer P1. For lysis 200 µl of P2 buffer was added and mixed by inverting the tube 5-6 times, the incubation was conducted by 30 sec at RT. The sample was neutralized with 200 µl of P3 buffer and mixed by inverting 4-5 times. After centrifugation for 15 min at 14,000x g, the supernatant was pipetted into a new Eppendorf tube and centrifuged again under the same conditions. The DNA in the supernatant was precipitated by adding 3 volumes of ice cold 100% ethanol and pelleted by centrifugation at 14,000x g for 15 min. The DNA pellet was washed with 500 µl of 70% ethanol and centrifuged again for 5 min. Then the pellet was dried and resuspended in 30 µl of Ampuwa H2O.
Buffer P1 (Resuspention buffer) 50 mM Tris/HCl, pH 8.0 10 mM EDTA
100 µg/ml RNase A Buffer P2 (Lysis buffer) 200 mM NaOH
1% (w/v) SDS
Buffer P3 (Neutralization buffer) 3 M Potassium acetate, pH 5.5
2. Material and Methods
2.2.1.2 Midi-Preparation of Plasmid DNA
High quality plasmid DNA was prepared with Anion-Exchange Resin columns from QIAGEN (Hilden) Plasmid Midi kit. For a midi preparation, 100 ml of an overnight culture (in LB medium plus antibiotic) was centrifuged at 4,000x g for 10 min. The bacterial pellet was resuspended in P1 buffer containing 100 µg/ml of RNase A. After lysis (P2 buffer) and neutralisation (P3 buffer), the suspension was recentrifuged at 13,000x g for 15 min.
The supernatant containing the DNA was applied to a previously equilibrated column (with QBT buffer), allowed to run by gravity and to bind the DNA to the anion exchange matrix.
The DNA was then purified from metabolites in 2 washing steps with buffer QC and eluted with an elution buffer (QF) and isopropanol was added followed by centrifugation at 13,000x g, 4°C for 30 min to pellet DNA. The pellet was dried, resuspended in Ampuwa H2O and stored at –20°C until use.
Buffer QBT (Equilibration buffer) 750 mM NaCl
50 mM MOPS, pH 7.0 15 % isopropanol (v/v) 0.15% Triton X-100 (v/v) Buffer QC (Wash buffer) 1.0 M NaCl
50 mM MOPS, pH 7.0 15 % isopropanol (v/v) Buffer QF (Elution buffer) 1.25 M NaCl
50 mM Tris/HCl, pH 8.5 15 % isopropanol (v/v)
2. Material and Methods
2.2.1.3 Isolation of Genomic DNA from Tissue Samples (Laird et al., 1991)
1.5 to 2.0 cm of the mouse tail was incubated in 700 µl of lysis buffer I at 55°C overnight in a thermomixer. To the tissue lysate, and equal volume of phenol was added, mixed by inverting several times and centrifuged at 8000xg at room temperature for 5 min. After transferring the upper aqueous layer into a new tube, the same procedure was repeated, first with 1:1 ratio of phenol and chloroform and then with chloroform. Finally, the DNA was precipitated with 0.7 volume of isopropanol, washed with 70% ethanol, and dissolved in 100-200 µl of TE buffer or H2O (Ampuwa) and incubated at 60°C for 15 min.
Lysis buffer I 100 mM Tris/HCl (pH 8.0) 100 mM NaCl
100 mM EDTA
0.2 mg/ml Proteinase K 0.5% SDS
2.2.1.4 Isolation of Genomic DNA from Cultured Blastocysts
Culturing of blastocysts is described in 2.2.21.3. After culturing, DNA was prepared by incubating the individual embryos with 20 µl of lysis buffer II for 4 h at 60oC followed by incubation at 90oC for 30 min. Three microliters of embryonic DNA was used for PCR.
Lysis-buffer II 50 mM Tris/HCl (pH 8.0) 0.5 mM EDTA (pH 8.0) 0.5% Tween 20
0.2 mg/ml proteinase K
2. Material and Methods
2.2.1.5 Preparation of Genomic DNA from Preimplantation Embryos
Embryos were obtained from superovulated and plugged female mice (2.2.21.1) at the following stages: 1-cell (18-20 hrs p.c.), 2-cell (42-44 hrs p.c.), 4-cell (50-52 hrs p.c.), 8 cell (66-72 hrs p.c.) morula and blastocysts (90-92 hrs p.c.). Individual embryos were collected in a PCR tube (0.2 ml) containing 5 µl Ampuwa H2O. Samples were repeatedly (2-3 cycles) frozen and thawed. Then samples were directly used for genotyping by using PCR approach.
2.2.1.6 Isolation of Total RNA from Tissue Samples
In order to avoid any RNase activity, all the equipment and solutions used for RNA isolation were previously treated with RNase AWAY and DEPC water. Special RNase free Eppendorf cups were used during the procedure. In this method, RNA is extracted in a denaturing solution of Guanidine-Isothiocyanate (GITC). Adult mice tissues (brain, liver, lung, kidney, ovary, testis, spleen, heart) were collected and used immediately for the RNA isolation. All of the collected tissues were separately prepared. 100 mg of each tissue was mixed with 1 ml of the GITC buffer solution (Total-RNA solution, Biomol) and then homogenized for 10-20 sec with an electric homogeniser (Ultraturrax T25, Schütt). Then 0.2 volume of cold chloroform was added and mixed. The samples were placed on ice for 5 min. After centrifugation of the suspension at 3.200x g for 10 min at 4oC, the upper phase was taken and mixed with 1 volume of ice-cold isopropanol and placed on ice for 15 min.
Then the RNA was precipitated by centrifugation at 13.000x g at 4°C and the pellet was washed with 1 ml of ice-cold 75% ethanol. The pellet was dried at room temperature (RT) and finally resuspended in 50-100 µl of DEPC-H2O. To avoid protein contamination, the samples were incubated at 65°C for 0.5-2 min, cooled on ice and the denatured proteins were pelleted by centrifugation for 30 sec. The RNA aliquots were stored at –80°C. The RNA concentration was determined with a spectrophotometer.
2.2.1.7 Phenol-Chloroform Extraction
One volume of phenol was added to the sample, vortexed and centrifuged at 13,000x g for
2. Material and Methods
was added. After vortexing and centrifugating as before, the supernatant was transferred to a fresh reaction tube for ethanol precipitation.
2.2.1.8 Ethanol Precipitation of Nucleic Acids
To precipitate nucleic acids, the salt concentration of the sample was adjusted to 300 mM with NaCl or with 0.1 volume of 3 M NaAc (pH 4,8) and 2.5 volume of 100 % ethanol was added. The sample was placed at -80°C for 20 min and pelleted by centrifugation (13,000x g, 15 min, 4°C). The pellet was washed with 70 % ethanol, centrifuged, dried and solved in an appropriate volume of H2O. The sample was then checked electrophoretically.
2.2.2 Enzymatic Modifications of DNA
2.2.2.1 Dephosphorylation of 5' Ends of DNA
The dephosphorylation of the 5' ends of DNA prevents the recircularisation of a previously restricted vector with compatible cohesive ends during a ligation experiment. This was achieved with the alkaline phosphatase (CIP from calf intestine), which removes the 5' phosphate from the free ends of DNA molecules. Therefore, the efficiency of the ligation of foreign DNA into a vector can be greatly enhanced. This treatment of the DNA was carried out as follows:
1 µg vector DNA
1 µl CIP, alkaline phosphatase (5 U/µl) 1 µl 10x CIP buffer
up to 20 µl Ampuwa dH2O
The reaction was performed at 37°C for 1 h and was terminated by heat inactivation at 68°C for 15 min and addition of 0.1 volumes of 10% (w/v) SDS and 0.1 volume of 10x TE buffer. After a phenol/chloroform extraction, the DNA was ethanol precipitated with 0.1 volume of 3 M NaAc (pH 4.8).
2. Material and Methods
10 x CIP buffer: 500 mM Tris/HCl, pH 9.0 10 mM MgCl2
1 mM ZnCl2 10 mM Spermidine 2.2.2.2 Cloning of DNA Fragments
Foreign DNA fragments were subcloned into vectors after digestion with restriction enzymes, electrophoresis separation (2.2.6.), isolation (2.2.7.) and finally ligation into a vector. The vector was restricted with one or two enzymes in the multicloning site (MCS).
When one enzyme was used to restrict the vector, a dephosphorylation step of the 5´ends (2.2.2.1.) was necessary to prevent the religation of the vector. The ligation reaction was done as follows with an overnight incubation at 16oC:
0.5 µg vector DNA
1-10x molar ratio of foreign DNA fragment 1 µl T4 DNA ligase enzyme (5U/µl) 1 µl 10x ligation buffer
up to 10 µl Ampuwa H2O
2.2.2.3 Cloning of PCR Fragments
PCR products (2.2.5.) were subcloned through the TA-ligation (TA: thymidine-adenine), if PCR was performed with a Taq polymerase that leaves a deoxyadenosine at the 3´end of the amplified fragment in the elongation phase of the PCR cycle. The PCR fragments were TA-ligated to the pGEM-T Easy vector (Promega). This vector contains an open insertion site inside the MCS that has a 3´overhanged thymidine where the PCR fragment ligates.
The ligation reaction was done as described in 2.2.2.2.
2. Material and Methods