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LifeTech Joint Project 2016 Laboratory manual Molecular Biotechnology group http://chemtech.aalto.fi/en/research/molecular_biotechnology/ 1 Table of Contents 1.1 Episomal genetic elements ............................................................................................... 4 1.2. Homologous recombination .............................................................................................. 5 1.3 Selection........................................................................................................................... 6 2. Health & Safety ........................................................................................................................ 7 3. 4. Yeast as an expression system ............................................................................................ 7 Strains ................................................................................................................................... 10 5. Protocols ................................................................................................................................... 11 5.1. Plasmid purification from E. coli .......................................................................................... 11 5.2. Protocol for PCR................................................................................................................. 13 5.3. Purification of DNA from agarose gels ................................................................................ 14 5.4. LiAc transformation of yeast from liquid culture .................................................................. 16 5.5. SDS-PAGE ......................................................................................................................... 18 5.6. Preparation of Western Blot Samples ................................................................................. 20 5.7. SDS PAGE and Western blotting ........................................... Error! Bookmark not defined. 5.8. ELISA: Enzyme-linked immunosorbent assay .................................................................... 21 5.9. Preparation of competent E. coli cells ................................................................................. 23 5.10. Transformation of E. coli ................................................................................................... 25 5.11. Colony PCR...................................................................................................................... 26 5.12. Gel electrophoresis........................................................................................................... 27 5.13. Ligation ............................................................................................................................. 29 5.14. Plasmid isolation ............................................................................................................... 30 6. Media ................................................................................................................................. 31 7. Buffers and solutions................................................................................................................. 33 2 3 1.1 Episomal genetic elements As indicated above, yeast cells have become popular due to their ease of genetic manipulation. A very convenient feature of S. cerevisiae is its ability to maintain genetic information on self-replicating episomal DNA elements or plasmids, like bacteria. A naturally occurring plasmid in S. cerevisiae, the so-called 2-micron plasmid, was used to construct the first E. coli-yeast shuttle vectors. These vectors contain sequences (origin of replication, selection marker) for propagation both in E. coli and S. cerevisiae and transform at high frequency (Fig. 3). Plasmids derived thereof are called YEp (Yeast episomal plasmids). These are maintained in a high copy number (20-50 copies) per cell and quite stably transmitted from mother to daughter cell but only in [cir+] strains carrying the natural endogenous 2-micron plasmid. Low copy number shuttle vectors, which are faithfully transmitted to the progeny, carry, instead of the 2-micron sequences, yeast chromosomal origins of replication, so-called autonomous replication sequences (ARS), and centromer sequences (CEN). Latter sequences are, in contrast to other fungi and higher eukaryotes, very compact (app. 150 bp) in S. cerevisiae. Such ARS-CEN-vectors or YCp (Yeast centromeric plasmids) are maintained in 1-5 copies per cell. Both YEp and YCp have a size of app. 5 kb and can accommodate up to 10-15 kb of additional DNA. For larger DNA fragments e.g. for genomic DNA libraries, yeast artificial chromosomes (YACs), which contain in addition to ARS and CEN telomeric sequences (TEL) and are linearized before transformation into S. cerevisiae, are available. Fig. 3. Scheme of E. coli yeast shuttle vectors. 4 1.2. Homologous recombination In contrast to most other organisms including other fungi, recombination of transformed DNA in yeast proceeds exclusively via homologous recombination. This special feature of S. cerevisiae can be exploited for various purposes. The observation that homologous recombination in yeast can be efficiently achieved using linear DNA has led to the rapid development of powerful methods for DNA manipulation. One method is the cloning of DNA fragments by in vivo recombination. In this case, a DNA fragment whose ends are homologous to the ends of a linearized shuttle vector can be directly recombined in yeast, alleviating the need for an in vitro ligation reaction (Fig. 4). Corresponding DNA fragment and linearized shuttle vector are cotransformed into S. cerevisiae and transformants are selected based on the yeast selection marker on the vector. The cells will form colonies only upon recombining the DNA fragments to a functional self-replicating stable plasmid. For amplification and further analysis, the plasmid is isolated from yeast cells and transformed into E. coli. The minimal length of the recombinogenic end is only 25 bp. This is short enough to be built into a PCR primer. This cloning by in vivo recombination in yeast is very precise and independent of restriction sites (except for the linearization site of the vector). Fig. 4. Cloning by in-vivo recombination in yeast. A DNA fragment, generated e.g. by PCR, with homologous ends is transformed into yeast together with a linearized shuttle vector. Homologous in vivo recombination between the vector and the DNA fragment results in a circular plasmid containing the DNA fragment, which can efficiently propagate. A second type of application is the targeted integration of exogenous DNA into the yeast chromosome. Depending on the design of the recombinogenic DNA, the exogenous DNA is either inserted at a specific location of the genome (so-called 'knock-in') or the recombination leads to replacement of a specific genome sequence by the exogenous DNA (so-called 5 'knock-out'). These applications allow genetic alterations such as gene replacements, promoter exchanges or insertion of epitope tags on the chromosome in single copy. Such chromosomal alterations have contributed significantly towards understanding the function of genes and proteins in vivo. A number of commercially available tools, such as a collection of knock out strains in all nonessential genes, facilitate such studies. Fig. 5. Gene replacement by homologous recombination A specific yeast open reading frame (ORF) is replaced by a linear DNA fragment carrying homologous flanking regions (red) and a marker gene coding for G418 resistance (kanMX4). The recombination leads to deletion of the yeast ORF, a process called gene knock out. 1. 1.3 Selection In bacteria, resistances against antibiotics are usually used as selection markers. In S. cerevisiae selection is usually based on amino acid or nucleotide biosynthetic genes in combination with respective S. cerevisiae mutants which are no longer capable of synthesizing the respective compound. The auxotrophic strains to be transformed are usually grown in complete medium and transformants are selected on minimal media lacking the respective compound. Usual selection markers are URA3 (Uracil biosynthesis), LEU2 (Leucine biosynthesis), HIS3 (Histidine biosynthesis) and TRP 1 (Tryptophan biosynthesis), and common laboratory S. cerevisiae strains such as SS328 (MATα ade2-101 his3Δ200 lys2-801 ura3-52) are mutant for several of these genes. Alternatively, dominant selection markers against a variety of antifungal drugs are available. Usually, these markers consist of the coding sequence of bacterial resistance genes under control of a strong, constitutive yeast promoter and terminator. Common dominant markers are resistances against Hygromycin (HYG), Neomycin (= G418, an antifungal Kanamycin derivative) and 6 Nourseothricin (NAT). In addition to these so-called positive selection procedures (growth requires uptake of DNA), there are also several negative or counterselection procedures available for S. cerevisiae (growth requires loss of DNA). The most often used counterselection is based on the fact that URA3 cells are sensitive to the drug 5-fluoroorotic acid (5-FOA) whereas ura3 cells are not. The non-toxic compound 5-FOA is thereby converted into the toxic compound 5-fluorouracil by the URA3 encoded enzyme. Such a counterselection can be exploited for various purposes. 2. 2. Health & Safety During the course, students will work with, and create, genetically modified organisms. All the included experiments can be carried out in a biosafety level 1 laboratory. During practical work in the laboratory, it is mandatory for students to wear a lab coat, safety goggles and protective gloves. Also, good care needs to be taken to ensure a clean workplace, and problems or a spilling should be reported immediately. Some of the experiments may include strong acids as reagents and chemical mutagens. Special care is required for working with Ethidium Bromide, which is a dangerous carcinogenic. Also during handling of the scalpels used for gel cut-outs, students need supervision and scalpels should be disposed immediately after usage. 3. Yeast as an expression system Outline of experiment Yeast has been widely used as an expression system for the production of heterologous proteins. A lot of factors involved in the expression and secretion process have been addressed in research, like expression levels, protein folding, vesicular transport, secretion efficiency, and degradation. In eukaryotes secreted proteins are targeted initially to the endoplasmic reticulum (ER). In contrast to prokaryotes, the eukaryotic cell has separate and specialized compartments of which the ER is designed for protein folding and sorting. Protein synthesis occurs in the cytosol but for many proteins the relocating into ER starts already during translation. After 7 passing the ER membrane through the protein translocation complex, the nascent polypeptide encounters multiple aspects that facilitate the protein to achieve its native conformation. The ER harbors an oxidative environment and a diverse set of folding factors which together enhance the rate in which the protein acquires its structural characteristics dictated by the amino acids sequence. Only correctly folded proteins will exit the ER and be secreted outside the cell. There are three major categories of folding factors: molecular chaperones, protein disulfide isomerases (PDIs), and peptidyl-prolyl cis-trans isomerases (PPIases). The ER has its own set of molecular chaperones that are distinct from cytosolic chaperones albeit their mechanism of action is the same. Chaperones bind to the hydrophobic patches of the polypeptide preventing the protein from aggregation. In the course of their action, chaperones bind and hydrolyze ATP. The hydrolysis and exchange of nucleotide (ADP to ATP) can be facilitated by additional proteins called co-chaperones, which speed up the binding cycle of the corresponding chaperone. PDIs on the other hand catalyze the formation and mixing of disulfide bonds, which form between two cysteine residues. Disulfide bonds are common in secreted proteins as these covalent bonds stabilize the native conformation in the varying extracellular environment. In the case of disulfide bonds, the oxidative environment of ER becomes important as it favors the formation and stabilization of the bonds. PPIases target certain prolines in the polypeptide: the backbone atoms of this amino acid can occur also in cis-configuration (naturally around 10 %) instead of the common trans-bond. This property is important in some proteins and switching from the trans to cis is catalyzed by PPIases. After the peptide backbone has achieved cisconformation, the folding protein is freed from the angle strain caused by unfitting backbone conformations. Although yeast is successfully used to produce and secrete insulin (by Novo Nordisk), other proteins are much more recalcitrant for expression in yeast. And one of the most difficult protein to express in yeast is the full-length antibody. IgG-antibodies are tetrameric, 150-kDa proteins that are secreted by the plasma cells of the mammalian immune system to fight against disease-causing agents. The subunits of IgG enter the ER separately, in which the complete protein is assembled. The folding of IgG employs all types of folding factors: chaperones, PPIases, and PDIs, which catalyze both the formation of inter- and intrachain disulfide bonds. The light chains and heavy chains do not fold separately but require interaction with each other to complete the process. Assembly 8 of the IgG-tetramer is catalyzed sequentially by the different folding factors as shown in Figure 13. Figure 13: The folding process of IgG. Figure is from Braakman and Bulleid 2011. References Gueldener et al, 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids res. 24, 2519-24. Feige et al, How antibodies fold. Trends Biochem Sci 35:189-198. Braakman and Bulleid, 2011. Protein folding and modification in the mammalian endoplasmic reticulum. Annu Rev Biochem. 80:71-99. 9 4. Strains In this experiment, the Saccharomyces cerevisiae W303α (leu2-3,112 trp1-1 can1-100 ura31 ade2-1 his3-11,15) strain is used. During the course of the experiments we will modify the yeast in order to improve expression of an antibody and analyze the outcome of the genetic engineering using different analytical methods. 3. 10 5. Protocols 5.1. Plasmid purification from E. coli 11 12 5.2. Protocol for PCR I. Create the following mixture for the PCR reactions: Knockout Colony 5X Phusion HF Buffer 10 µL 2 µL 10mM dNTPs 1 µL 0,2 µL Primer 1 2.5 µL 0,5 µL Primer 2 2.5 µL 0,5 µL Template DNA 1,5 µL (plasmid 6,7 µL (boiled pUG6) cells) ddH2O 32.0 µL 0 µL Phusion Polymerase 0,5 µL 0,1 µL Total 50 µL 10 µL IMPORTANT: Make sure that the Phusion polymerase is added lastly and that it is kept in the Dynafreezer box when it is out of the freezer! a. Put the samples in the thermocycler and run the following programme: Knockout Colony I. Initial denaturation 98 C 30 s 98 C 30 s II. Denaturation 98 C 10 s 98 C 10 s III. Annealing 54 C 30 s 65C 30 s IV. Extension 72 C 45 s 72 C 45 s 6x back to 2 V. Denaturation 98 C 10 s VI. Annealing 68 C 30 s VII. Extension 72 C 45 s 36x back to 2 29x back to 5 VIII. IX. II. Final extension 72 C 10 min 72 C 10 min Cool samples 4C Pause 4C Pause When the PCR is finished, adjust the samples with 6X DNA sample buffer and load the samples on the gel. Run at 90V until separated sufficiently. 13 5.3. Purification of DNA from agarose gels 14 15 5.4. LiAc transformation of yeast from liquid culture Yeast cells treated with alkali metal ions are able to uptake linear and circular DNA molecules from their surroundings. The LiAc-procedure is fast and easy and it doesn’t require disruption of the cell wall. The following is a common protocol for routine manipulation of yeast cells where the transformation efficiency is up to 10 3 cfu per µg of DNA. The DNA to be transformed is supplied with single stranded carrier DNA (from salmon sperm) to stabilize and enhance the transformation process. Solutions 10x TE buffer pH 7.5 1M LiAc 50 % PEG-4000 DMSO Solution A: For 10 mL: 1mL 10x TE, 1mL 1M LiAc, 8 mL ddH2O Solution B: For 10 mL: 1mL 10x TE, 1mL 1M LiAc, 8 mL 50% PEG-4000 Yeast Cultivations Inoculate YPD main culture with saturated overnight culture to reach a start OD600 of 0.5 and incubate at 30° C until an OD600 of at most 2.0 is reached, takes about 3 hours. You need 5 ml of culture per transformation reaction. Preparing competent cells Harvest cells by centrifugation at 3900 rpm for 5 minutes RT (use Falcon tube) Decant supernatant (collect to microbial waste) Resuspend pellet with 30ml sterile H2O Repeat centrifugation as described above After decanting supernatant, resuspend pellet in 500 μl solution A (1x TE, 0.1% LiAc) and move to an Eppendorf tube. Spin cells down (3 min at 3600 rpm in an Eppendorf centrifuge) Per assay you'll need 100 μl suspended cells, so resuspend pellet in 100-300 μl solution A (1xTE, 0.1% LiAc) 16 Transformation of LiAc competent cells Add 1-3 μl (600ng) of each plasmid DNA (the purer the DNA the less you need) Mix plasmids with 3 μl salmon sperm [10 μg/ml], heat denatured (5 min at 100 °C) as carrier DNA Add 100 μl cells (in solution A) to the tube Add 1 ml solution B (1x TE, 0.1% LiAc, 40% PEG) and mix by inverting tube (no vortexing!) Incubate for 1 h at 30 °C Add 120 μl DMSO (invert tube to mix) Heat shock cells for 10 min at 42 °C in a water bath Cool down cells immediately on ice for 3 min Spin down cells for 5 min at 3000 rpm in an Eppendorf centrifuge Remove supernatant by pipetting Resuspend pellet carefully in 100 µL ddH2O Plate cells on selective medium and incubate at 30 °C (first transformants should be visible after 2 days). When transforming cells with a knockout cassette up to 500 ng of DNA should be transformed. After heat shock, cells are resuspended into 1 mL of YPD (or other nonselective medium) and incubated at 30˚C for at least 3 hours. Then plate on selective media 17 5.5. SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins according to their molecular weight. Note that unpolymerized acrylamide and TEMED are poisonous, try not to inhale and preferably use in fume hood. Materials: 10 % APS TEMED 40 % acrylamide 4x Separating buffer (1.5 M Tris-HCl pH 8.8, 0.4 % SDS) 2x Stacking gel (0.25 M Tris-HCl pH 6.8, 0.2 % SDS) sterile H2O Casting the gel 1. Check that the glasses are clean. Wash and rinse with water and ethanol if necessary. 2. Set up the casting gear. Press glasses against each other so that the lower end is even (check by placing on the table for example). You may check with water that the set-up doesn’t leak. Remove the water by pouring and Whatman filter paper. 3. Mark the border of separating and stacking gel by measuring 0.5 cm below the comb. 4. Mix the separating gel according to the table below. The mixture is enough for two gels. APS and TEMED are responsible for the polymerization so add these last. acrylamide 4x end separating H2O (ml) concentration buffer (ml) 40 % 10 % APS TEMED Acrylamide (µl) (µl) (ml) % 7.5 2.5 5.625 1.875 60 12.5 12.5 2.5 4.375 3.125 60 12.5 5. Pour or pipette the mixture between the glasses till the marking. 6. Add 500 µl saturated butanol or isopropanol evenly on top of the gel. 7. Let the separating gel solidify (takes around 45 minutes). 8. Mix the stacking gel according to the scheme. a. The amounts below are for four gels. 18 acrylamide 2 x stacking end buffer (ml) H2O (ml) concentration 40 % 10 % APS TEMED Acrylamide (µl) (µl) 60 12.5 (ml) % 3 3.8 3.2 0.6 9. Pour butanol/isopropanol out and rinse with ethanol/water. Dry with Whatmanpieces. 10. Add the stacking gel –mixture on top of the gels. Push the combs inside and let the stacking gel solidify (for about 20 minutes). 11. You can use the gel for running right away or store it at +4 ˚C wrapped in moist paper and plastic/aluminium foil. 19 5.6. Preparation of Western Blot Samples The content of Western blot samples is basically the same as prepared for normal SDSPAGE. However, the specificity and sensitivity of Western blotting enables that higher amounts of protein can be loaded to the gel to detect and quantify proteins that are present in low amounts. In the first protocol, the cells are destroyed to extract the intracellular proteins for analysis. In the course of treatment, protease inhibitor should be used as intracellular proteases can degrade the target molecule. For supernatant samples, acetone precipitation is used to concentrate proteins in the sample. This enriches the produced antibody in such degree that it can be easily seen on a blot. Cell extract preparation NOTE: To avoid sample degradation it is important to keep the samples on ice during this protocol! 1. At the designated time point the culture is transferred to a falcon tube and put on ice 2. After OD600 measurement the cells are spun down for 5 minutes, 3900 rpm, + 4 ˚C 3. The supernatant is collected to a separate tube and the cell pellet is resuspended to a concentration of 0.05 OD/µl in ddH2O 4. 200 µl of suspension is transferred to an eppendorf tube and spun down for 5 minutes, 5000 rcf + 4 ˚C. 5. The pellet is resuspended in 200 µL sample buffer without reducing agent with 1X protease inhibitor and 1 mM PMSF. 6. Half of the volume of acid washed glass beads is added. 7. Samples are vortexed for 10 minutes at full speed in the cold room. 8. To remove foam, the samples are spun down at full speed for 5 minutes + 4 ˚C. 9. Take out supernatant (around 100 µl) to a new Eppendorf. Do also reduced samples by taking 47,5 µl supernatant and mixing it with 2,5 µl 1 M DTT. 10. The samples are boiled for 5 minutes at 65 ˚C. 20 5.7. ELISA: Enzyme-linked immunosorbent assay Solutions: PBS PBT (PBS with 0.05 % Tween-20) Coating: Goat anti-human IgG (SIGMA, #2316) Fc specific 2.2 mg/mL. Working solution is made by diluting 19,1 µL in 10 mL PBS Standard: human IgG (Myeloma) 2.93 µL in 10mL PBT yields 1 µg/mL. Make serial 1:2 dilutions in 10 mL volumes to the following concentrations: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625 and 0.0078125 µg/mL Detection antibody: Goat anti-human IgG peroxidase conjugated (SIGMA, #A0170) Fc Specific 7,1 mg/ml. Diluted 1:4000 in PBT (3 µl per 12 ml) Substrate: 200 µL of a 10 mg/mL ortho-phenylenediame (OPD) is diluted in 10 mL of 0.05M phosphate-citrate buffer pH 5, shortly before use 3µL of 30% H2O2 is added. o Note: be careful as OPD is toxic and collect all OPD-containing waste in the designated container! 3 M H2SO4 (for stopping the reaction) Protocol with 96-well plates: 1. Wells are coated with 100 µl goat anti-human IgG (4.2 µg/ml in PBS) per well by incubation overnight at 4 ˚C on a shaking platform. 2. The next day, the plate is washed with 200 µl PBT per well and then removing the liquid, repeat 5 times. 3. For blocking the plate is incubated with 200 µl PBT per well for 45 minutes at RT on a shaking platform, the plate is covered with a lid or foil. 4. The plate is washed with 200 µl PBT per well and then removing the liquid, repeat 5 times 5. Standards and samples are added 200 µl per well according to pipetting schema and incubated for 1.5 hour at RT. 6. The plate is washed with 200 µl PBT per well and then removing the liquid, repeat 5 times. 21 7. Detection antibody is added 100 µl per well and incubated for 1 hour at RT on a shaking platform; the plate is covered with a lid or foil. 8. The plate is washed with 200 µl PBT per well and then removing the liquid, repeat 5 times. 9. Substrate is added 100 µl per well and incubated for 8 minutes. Each row is pipetted with 10 seconds intervals to ensure same incubation time for each well. 10. Reaction is stopped by addition of 100 µl 3 M H2SO4 per well, with the same 5 seconds intervals. 11. The signal is measured on plate reader at 450nm. 22 5.8. Preparation of competent E. coli cells !! All steps from harvesting the cells should be carried out at 4 °C and the cells should then be kept on ice whenever possible! Make sure the CaCl2, CaCl2+25% glycerol and Eppendorf tubes are pre-chilled as well. !! Materials: E. coli cells (from a plate or -80°C competent stock) – Top10, XL-blue, DH5α LB medium (sterile) 0.1 M CaCl2, ice cold (sterile) 0.1 M CaCl2+25% glycerol, ice cold (sterile) A lot of 1.5 mL Eppendorf tubes, ice cold (sterile) 1. (Optional) Inoculate strain of E. coli into 3-5 mL LB and grow O/N at 37°C + shaking. 2. Next morning, use 1 mL to make a 200 ml liquid culture of cells (LB, in a 1L erlenmeyer flask). Grow the cells at 37°C + shaking (~220 rpm) until the OD600 is between 0.3 – 0.4 (usually takes around 2 hours). Do NOT let the OD600 get > 0,5!! 3. Pour the culture into 4x 50mL Falcon tubes and chill the cells on ice for 15-30 min. 4. Centrifuge the cells for 10 min at ~3300 g (aka ~4500 rpm for the Eppendorf 5804R tabletop centrifuge) at 4 °C. 5. Discard ALL the supernatant. Add 50 mL of 0.1 M CaCl2 to each tube and resuspend cells. 6. Incubate the cells on ice for at least 30 min (up to 3 hours). 7. Centrifuge the cells for 10 min at ~3300 g at 4 °C. 8. Discard supernatant and resuspend each cell pellet in 1 mL of 0.1 M CaCl 2+25% glycerol. 9. Aliquot cells into pre-chilled, sterile eppendorfs (e.g. 100 µL per tube). 10. Snap-freeze aliquots with liquid nitrogen and store in -80°C freezer. Notes & Hints: Chemically competent cells have lower transformation efficiencies than with electroporation, but are simpler to transform, less work to make and have the advantage 23 that larger volumes of DNA containing buffers etc (e.g. after Golden Gate cloning) can be transformed more easily. After making competent cells, the transformation efficiency should be checked with a known plasmid of known concentration. e.g.: To calculate transformation efficiency, transform 2 μl of the provided control pUC19 DNA (100 pg) into 50 μl of cells. Follow regular transformation protocol and dilute 10 μl of cells up to 1 ml in SOC. Plate 100 μl of this dilution. In this case, 150 colonies will yield a transformation efficiency of 1.5 x 109 cfu/μg (μg DNA=0.0001, dilution=10/1000 x 100/1000). For good transformation results, the transformation efficiency of the competent cells should be ~1 x 10 9 colony forming units (cfu) per μg. (source: NEB Q5 SDM) Step 5: When resuspending cells, first add 25 mL, vortex, shake, pipet up and down, then add remaining 25 mL. Step 8: Doesn’t matter if there is still a bit supernatant left because of the high amount of glycerol used. You can also add 1.5 or 2 mL instead, for more tubes but with lower cell concentration. Step 9: Aliquot amount suited for 1 or a few transformations, e.g. 100 or 200 µL per tube. For least work: pre-chill tubes in cold room, and put all of them in a labeled ziplock bag (no need to label tubes individually). Step 10: Snap-freezing can be skipped. 24 5.9. Transformation of E. coli Transforming chemically competent cells using heat shock 1. Pipet 50 µL of competent cells and 2 µL of DNA (or ligation reaction) into an eppendorf tube, mix gently 2. Incubate tubes on ice for 30 minutes 3. Incubate tubes in 42 °C for 1 minute 4. Incubate tubes on ice for 5 minutes 5. Add 500 µL of SOC 6. Incubate cells at 37 °C for 1 hour 7. Collect cells by centrifugation and remove 450 µL supernatant 8. Resuspend cells in remaining supernatant and plate onto an appropriate antibiotic LBagar plate 9. Incubate overnight at 37 °C 25 5.10. Colony PCR 26 5.11. Gel electrophoresis Preparing the agarose gel For other percentages adjust amounts and volumes accordingly! 1. Measure 1.25 g Agarose powder and add it to a 500 ml flask 2. Add 125 ml TAE Buffer to the flask. (the total gel volume well vary depending on the size of the casting tray) 3. Melt the agarose in a microwave or hot water bath until the solution becomes clear. (if using a microwave, heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask). 4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. 5. ADD 5 µl EtBr to the solution 6. Seal the ends of the casting tray with two layers of tape. 7. Place the combs in the gel casting tray. 8. Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white). 9. Carefully pull out the combs and remove the tape. 10. Place the gel in the electrophoresis chamber. 11. Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel. 12. Note – gels can be made several days prior to use and sealed in plastic wrap (without combs). If the gel becomes excessively dry, allow it to rehydrate in the buffer within the gel box for a few minutes prior to loading samples. Loading the gel 1. Add 5 l of 6X Sample Loading Buffer to each 25 l PCR reaction 2. Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder. 3. Carefully pipette 20 l of each sample/Sample Loading Buffer mixture into separate wells in the gel. 4. Pipette 5 l of the DNA ladder standard into at least one well of each row on the gel. Note – if you are running multiple gels, avoid later confusion by loading the DNA ladder in different lanes on each gel. 27 Running the gel 1. Place the lid on the gel box, connecting the electrodes. 2. Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”) 3. Turn on the power supply to about 100 volts. Maximum allowed voltage will vary depending on the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes! . 4. Check to make sure the current is running through the buffer by looking for bubbles forming on each electrode. 5. Check to make sure that the current is running in the correct direction by observing the movement of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the DNA). 6. Let the power run until the blue dye approaches the end of the gel. 7. Turn off the power. 8. Disconnect the wires from the power supply. 9. Remove the lid of the electrophoresis chamber. 10. Using gloves, carefully remove the tray and gel. 28 5.12. Ligation 29 5.13. Plasmid isolation 30 6. Media LB -Broth (Luria Bertani Broth) Bacto Yeast Extract 0.5 % 5.0 g Bacto Tryptone 1.0 % 10.0 g Natriumchlorid 0.5 % 5.0 g Agar (for plates) 1.5 % 15 g If necessary adjust pH to 7.2 with NaOH Adjust volume to 1000 ml with ddH2O and autoclave YPD Final concentration For 1 liter Yeast Extract 1.0 % 10.0 g Peptone (bacteriological) 2.0% 20.0 g Dextrose = Glucose 2.0 % 20.0 g Agar (for plates) 2.0 % 20.0 g If necessary adjust pH to 6 with 6 M HCl Adjust volume to 1000 ml with ddH2O and autoclave Mimimal Medium (for selection ofprototrophs) Yeast Nitrogen Base w/o amino acids 0.67 % Glucose 2% Agar (for plates) 2% Adjust volume to 1000 ml with ddH2O and autoclave. 6.7 g 20.0 g 20.0 g SD Medium (Synthetic Complete Dextrose) Mix C- and N-source from Minimal Medium (Nitrogen Base and Glucose) and add amino acids from the drop-out mix mentioned in list below. For selection simply leave out the respective amino acid 31 Drop-Out Mix (for selection of auxotrophic markers leave out the respective amino acid) 1x 2x Adenine 0.5 g 1.0 g Alanine 2.0 g 4.0 g Arginine 2.0 g 4.0 g Asparagine 2.0 g 4.0 g Aspartic acid 2.0 g 4.0 g Cysteine 2.0 g 4.0 g Glutamine 2.0 g 4.0 g Glutamic acid 2.0 g 4.0 g Glycine 2.0 g 4.0 g Histidine 2.0 g 4.0 g Inositol 2.0 g 4.0 g Isoleucine 2.0 g 4.0 g Leucine 4.0 g 8.0 g Lysine 2.0 g 4.0 g Methionine 2.0 g 4.0 g para-Aminobenzoic acid 0.2 g 0.4 g Phenylalanine 2.0 g 4.0 g Proline 2.0 g 4.0 g Serine 2.0 g 4.0 g Threonine 2.0 g 4.0 g Tryptophane 2.0 g 4.0 g Tyrosine 2.0 g 4.0 g Uracil 2.0 g 4.0 g Valine 2.0 g 4.0 g Use mortar and pestle to mix. Keep as powder! Use 2 g of the mixture for 1 liter medium 32 7. Buffers and solutions Final concentration For 1 liter 10x Running Buffer (pH ~ 8.3) Tris base 0.25 M 30.3 g Glycine 1.92 M 144.0 g SDS 1% 10.0 g Adjust volume to 1000 ml with ddH2O It is not recommended to adjust pH! If you add HCl to the buffer, the additional Cl- ions may interfere with the electrophoresis resulting in very long run-times. 4x Separating Gel Buffer (pH 8.8) Tris base 1.5 M 182.0 g SDS (20 % stock solution) 0.4 % 20.0 ml Adjust pH to 8.8 with HCl Adjust volume to 1000 ml with ddH2O 2x Stacking Gel Buffer (pH 6.8) Tris base 0.25 M 30.0 g SDS (20 % stock solution) 0.2% 10.0 ml Adjust pH to 6.8 with HCl Adjust volume to 1000 ml with ddH2O 4x Sample Buffer (Lämmli) for 40 ml 1M Tris-HCl (pH 6.8) 0.25 M 10.0 ml SDS 8% 3.2 g Glycerol (80%) 40 % 20.0 ml β-Mercaptoethanol 20 % 8.0ml Bromophenolblue 0.2 % 8.0 mg Adjust volume to 40 ml with ddH2O, mix well and aliquot, store at -20°C Western Blot Transfer Buffer (semi-dry blotting) For 1 liter 33 10x SDS Running Buffer 100 ml Methanol 200 ml ddH2O 700 ml PBS 20x (phosphate buffered saline) NaCl 2.7 M 160.0 g KCl 0.05 M 4.0g *Na2HPO4 0.2 M 28.8 g KH2PO4 0.35 M 4.8g Adjust volume to 1000 ml with ddH2O, autoclave, store at RT Usually, pH is around 8.3 when buffer is concentrated 20x. If you need a certain pH, first dilute 20x stock in 800 ml, then adjust pH to e.g. 7.4 with HCl or 10 % KOH and adjust volume to 1000 ml with ddH2O. TBS (tris buffered saline) Final concentration For 1 liter Tris-HCl (Tris base, adjust pH to 7.5 with HCl) 50 mM 6.1 g NaCl 150 mM 8.8 g PBS-T, TBS-T: PBS or TBS containing 0.1 % Tween 20 10x TE (100 mM Tris-HCl, 10 mM EDTA pH 8.0) For 10 ml Prepare 100 ml 1 M Tris-HCl (Tris base, adjust pH to 8.0 with HCl) 1.21 g Prepare 20 ml Na2EDTA (0.5 M stock pH 8.0), see recipe for preparing EDTA stock solution Mix 100 ml Tris-HCl 1 M pH 8.0 with 20 ml EDTA 0.5 M (pH 8.0) Adjust to 1000 ml with ddH2O, autoclave Store at room temperature 34 35