centriole disengagement
4. MATERIAL AND METHODS
4.3. Molecular biological methods
To produce chemically competent bacteria, 300 ml SOB medium were inoculated with 4 ml of an overnight culture of XL1 blue with an OD600 of 0.5. The culture was chilled on ice for 15 min and cells were harvested by centrifugation (4°C, 3000 g, 15 min). All following steps were performed at 4°C. The pelleted bacteria were carefully resuspended in 90 ml Tbf1 buffer and incubated on ice for 15 minutes. After another centrifugation (4°C, 1500 g, 15 min) the cells were resuspended in 15 ml Tbf2 buffer and placed on ice for 5 min. Aliquots of this suspension were snap-frozen in liquid nitrogen and stored at -80°C.
4.2.5. Transformation of E. coli
To transform E. coli with Plasmid DNA, chemically competent cells were thawed on ice. 50 µl of the suspension were carefully mixed with 50 ng of plasmid DNA or 5 µl of ligation mix (see 4.3.7) and incubated on ice for 20 min. After a heatshock at 42°C for 45 s the suspension was chilled on ice for 2 min, followed by addition of 500 µl LB medium and incubation at 37°C for 30 min to allow the ampicillin resistance gene to be expressed. To select transformed cells, the suspension was plated on LB agar containing ampicillin.
4.3. Molecular biological methods
4.3.1. Isolation of plasmid DNA from E. coli
2 ml LB medium supplemented with ampicillin were inoculated with a single colony of transformed E. coli. After incubation at 37°C in a rotator, plasmid DNA was purified by alkaline lysis followed by DNA purification via silica columns according to the manufacturer’s instructions (Fermentas/Thermo Scientific, Schwerte, Germany).
Higher amounts of plasmid DNA for transfection of human cells were isolated out of 50-100 ml of over night culture using a plasmid midi preparation kit (QIAGEN, Hilden, Germany).
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4.3.2. Determination of DNA concentrations in solutions
DNA concentrations in solutions were measured by determination of the optic density at a wavelength of 260 nm (OD260) using a ND-1000 spectrophotometer (PeqLab, Erlangen, Germany). An OD260 of 1 corresponds to 50 µg/ml double-stranded DNA.
4.3.3. Restriction digestion of DNA
All used restriction enzymes were obtained from either NEB (Frankfurt a. M., Germany) or Fermentas/Thermo Scientific (Schwerte, Germany) and digestion was performed according to the manufacturer’s instructions. Normally 1-5 units of enzyme were used to digest 1 µg DNA. Samples were incubated at the suggested temperatures for 1 h.
4.3.4. Dephosphorylation of DNA fragments
To prevent religation of digested vectors, the 5’-end of the vector DNA was dephosphorylated by addition of 1 unit of antarctic phosphatase (NEB, Frankfurt a.
M., Germany) in the corresponding buffer at 37°C for 1 h.
4.3.5. Separation and analysis of DNA fragments by agarose gel electrophoresis
TPE buffer 50 mM
0.13% (v/v) 2 mM
Tris H3PO4
EDTA
TBE buffer 90 mM
90 mM 2.5 mM
Tris boric acid EDTA 6x DNA loading buffer 50% (v/v)
0.1 M 0.02% (w/v) 0.02% (w/v) 0.02% (w/v)
glycerol EDTA
xylene cyanol bromophenol SDS
MATERIAL AND METHODS
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For analysis or for preparative isolation, DNA fragments were electrophoretically separated on agarose gels. For this purpose, 1-2% agarose gels were prepared with TPE buffer (separation of fragments > 1 kb) or TBE buffer (separation of fragments
<1 kb) and supplemented with ethidium bromide (final concentration: 0.5 µg/ml). The DNA samples were mixed with loading buffer (to 1x) and separated at 100 V in TBE or TPE buffer. The DNA fragments could be visualized due to the intercalation of ethidium bromide with the DNA using a UV transilluminator (324 nm). Size of the fragments was estimated using the standard size marker O'GeneRuler 1 kb DNALadder (Fermentas, St. Leon-Rot, Germany) or a self-made standard made from EcoRI-restriction digestion of SPP1 bacteriophage DNA.
4.3.6. Isolation of DNA from agarose gels
After gel electrophoresis, the band of the desired fragment was excised with a scalpel from the gel. The DNA was extracted using the GeneJet Gel Extraction Kit (Fermentas/Thermo Scientific, Schwerte, Germany) according to the manufacturer's instructions.
4.3.7. Ligation of DNA fragments 10x T4 DNA ligase buffer 50 mM
10 mM 1 mM 10 mM
Tris-HCl, pH 7.5 MgCl2
ATP DTT
The amounts of insert and linearized, dephosphorylated vector and were estimated on an agarose gel. For the ligation, the molar ratio of vector to insert was adjusted to 1:3. The reaction mix of 10 µl usually additionally contained 1 µl of T4 DNA ligase (self-made in the lab) and 1 µl ligase buffer and was incubated at room temperature for 1 h.
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4.3.8. Polymerase chain reaction (PCR)
For PCR reactions, a 50 µl mix containing 0.2 µl of 5'- and 3'- oligonucleotides (100 pmol/µl), 1 µl deoxyribonucleotide triphosphate mix (10 mM , NEB , Frankfurt a . M.) and 1 U DNA polymerase (Phusion, Fermentas/Thermo Scientific, Schwerte, German) in the corresponding PCR buffer (Phusion HF or GC buffer). As a template, 100 ng of plasmid DNA were used. Amplification was carried out in a TC-512 cycler (Techne, Burlington, NJ , USA).
step temperature duration number of
cycles
initial denaturation 98°C 1 min 1
denaturation 98°C 20 s
annealing optimized for used
oligonucleotides 20 s 25
elongation 72°C 30 s/kb
final elongation 72°C 5 min 1
4.3.9. Mutagenesis PCR
Site-directed mutagenesis was accomplished by a fusion PCR based method. To this end, two reverse-complement primers were used which carried the desired mutations. In two different PCR reactions using one of the mutagenesis primers an upstream and a downstream fragment generated. The respective outer primers were designed to carry additional restriction sites at their 5' ends. After gel purification, PCR was performed using the two PCR products (which anneal to and therefore prime each other) and the two outer primers to create a fused fragment. The resulting fragment was digested with the corresponding restriction enzymes and ligated into a identically digested vector carrying the wild-type ORF. The mutations were verified by sequencing (SeqLab, Göttingen, Germany).
4.3.10. Quantitative PCR (qPCR)
To quantify the amounts of SGO1 cDNA in different cancer tissues, I performed qPCR on commercial cDNA (brain, testis and fetal kidney, Takara/Clontech, Saint-Germain-en-Laye, France) and “TissueScan” cancer tissue cDNA arrays (Cancer survey I CSRT 101, OriGene, Rockville, MD, USA) containing 96 samples of cDNA,
MATERIAL AND METHODS
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which were obtained from 8 different tissues (breast, colon, kidney, liver, lung, ovaries, prostate and thyroid gland) at different stages of cancer. To quantify amounts of cDNA the TaqMan® system was used, which consists of a pair of unlabeled PCR primers and a probe with a fluorescent dye label on the 5’ end and non-fluorescent quencher (NFQ) on the 3’ end. During amplification, the polymerase reaches the probe and its endogenous 5’ nuclease activity cleaves the probe, separating the dye from the quencher. The following forward and reverse primers were used in combination with a probe that was coupled to a FAM (6-carboxyfluorescein) reporter at the 5’ end and a quencher (NFQ, non-fluorescent quencher) at the 3’ end (designed with the Applied Biosystems software) targeting the unique 3’ ends of either SGO1 A1 or SGO1 A2 and C2 cDNA).
name sequence
SGO1 A1_fwd 5’-CTGGGATTACTGAGCCACTGT-3’
SGO1 A1_rev 5’-TGCCAGAAGCTTATAATTAAAAGATCTTATTTGAGTA-3’
SGO1 A1_probe FAM-5’-CCCAAAATGTATCTTATACAAACAT-NFQ SGO1 A2/C2_fwd 5’-AGCCCTTTAATTAGAGATAGCAACTTTCC-3’
SGO1 A2/C2_rev 5’-CAATCTCCAAGTGACACAACCAAAA-3’
SGO1 A2/C2_probe FAM-5’-CTGATTCCTCGGTCACCC-3’-NFQ
To guarantee that identical amounts of template were used in all experiments, levels of SGO1 cDNA had to bee normalized to cDNA levels of housekeeping genes. For initial optimization of the qPCR protocol, duplex-qPCR was performed with either SGO1 A1 or A2 and C2-primer mix in combination with a commercially available GAPDH primer mix, containing forward and reverse primers against GAPDH cDNA and a probe, coupled to a VIC reporter at the 5’-end and NFQ at the 3’-end (TaqMan® Gene expression Assays, GAPDH, no 4448484, Applied Biosystems/Thermo Scientific, Schwerte, Germany). The cDNAs of the cancer tissue array that were custom-plated on two 46-well plates were already normalized to β-actin expression. The qPCR reaction mix prepared as follows:
TaqMan universal master mix (no. 4324018, Applied
Biosystems)
10 µl
primer mix SGO1 1 µl
primer mix GAPDH * 1 µl
cDNA * 0.5 or 1 µl
ddH2O ad 20 µl
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For expression studies of the cDNA array, the reaction mix was prepared without cDNA, vortexed, 20 µl were added to each well and the plates were sealed with adherent foil. The plates were then vortexed on the lowest speed, incubated on ice for 15 min, before being analyzed with a StepOne™ Real-Time PCR cycler (Applied Biosystems/Thermo Scientific, Schwerte, Germany).