Team:DTU-Denmark-2/Team/Protocols

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	Protocols 1 List of primers 2 Amplification of biobricks by PCR 2.1 PCR MIX 2.1 Procedure 2.2 PCR Programs 2.3 Purification of PCR Product 3 Gel electrophoresis 4 USER cloning 4.1 Tranformation in E.coli 4.2 Cultivation of transformed cells 4.3 Purification of plasmids 5 Fungi 5.1 Transformation in fungi 5.2 Production of conidiospores 5.3 Flourescence Microscopy 5.4 Restriction enzyme analysis 5.5 Fluorescence detection 5.5 Extraction of proteins 6 Assays 6.1 ß-galactosidase assay 6.2 Bradford assay 7 Mammalian cells 7.1 Cell culture and reagents 7.2 Medium 7.3 Cultivation of cells 7.4 Passing and maintenance of U2OS cells 7.5 Transferring cells to coverslips 7.6 Transfection of cells 7.7 Coverslides for microscopy 7.8 Purification of plasmides	Primer list for mammalian BioBricks and for fungal BioBricks Fungal primers Primer name Sequence pgpd FW ACGTCGCUATTCCCTTGTATCTCTACACACAGG pgpd RV ATCGCACUGCGGTAGTGATGTCTGCTCAA pAlc FW ACGTCGCUCTCCCCGATGACATACAGGAGG pAlc RV ATCGCACUTTTGAGGCGAGGTGATAGGATTG DMKP-P6 FW ACGTCGCUATCTCACTCCACTAGAATTCCTGTC DMKP-P6 RV ATCGCACUAAATGAGTCGAGAATGGCGG GFP-module FW AGTGCGAUATGGTGAGCAAGGGCGAG GFP-module RV ATCGGAAUTTACTTGTACAGCTCGTCCATGC GFP-GOI FW AGTGCGAUATGGTGAGCAAGGGCGAG GFP-GOI RV ATCGCTCUTTACTTGTACAGCTCGTCCATGC GFP-TS FW AGCGCTGGUATGGTGAGCAAGGGCGAG GFP-TS RV ATCGGAAUTTACTTGTACAGCTCGTCCATGC GFP-PTS1_module RV ATCGGAAU TTACAGCTTGGA CCTTGTACAGCTCGTCCATGCC RFP-module FW AGTGCGAUATGGCCTCCTCCGAGGAC RFP-module RV ATCGGAAUTTAGGCGCCGGTGGAGTG RFP-GOI FW AGTGCGAUATGGCCTCCTCCGAGGAC RFP-GOI RV ATCGCTCUTTAGGCGCCGGTGGAGTG RFP-TS FW AGCGCTGGUATGGCCTCCTCCGAGGAC RFP-TS RV ATCGGAAUTTACTTGTACAGCTCGTCCATGC RFP-NLS_module RV ATCGGAAU TTAGACCTTGCGCTTTTTCTTGGG GGCGCCGGTGGAGTGG LacZ FW AGTGCGAUATGACCATGATTACGGATTCACT LacZ RV ATCGGAAUTTATTTTTGACACCAGACCAACT trpC FW ATTCCGAUGATCCACTTAACGTTACTGAAATCA trpC RV ACGCAAGUGGGCGCTTACACAGTACACGAG PyrG FW ACTTGCGUCGTGGAGTTACCAGTGATTGACC PyrG RV AGCTTAAUCTTGCTAGATGACTGGTAGGAATCT bleR FW ACTTGCGUATGGCCAAGTTGACCAGTG bleR RV AGCTTAAUTCAGTCCTGCTCCTCGGCC hyrR FW ACTTGCGU GCTAGTGGAGGTCAACACATCAAT hyrR RV AGCTTAAUCGGTCGGCATCTACTCTATTCC argB FW ACTTGCGUCGCGGTTTTTTGGGGTAGT argB RV AGCTTAAUGCCACCTACAGCCATTGCGAA PyrG-DR FW ACTTGCGUTGGATAACCGTATTACCGCCT PyrG-DR RV AGCTTAAUTGCCAAGCTTAACGCGTACC ptrA FW ACTTGCGUGGAGATCGTCCGCCGATG ptrA RV AGCTTAAUCTAGAATGCCCCACCGTTACATAC Amp-cas FW AGCTTAAUTTACCAATGCTTAATCAGTGAGGC Amp-cas RV ACTTGCGUGACGTCAGGTGGCACTTTTCG yA FW AGTGCGAUATGTACCTCTCCACGGTCCTCT yA RV ATCGCTCUCTAAGAATCCCAAACATCAACCC MTS FW AGTGCGAUATGTTTACAGCGGCAGCTCG MTS RV ACCAGCGCUCTTGCGCCGCGGAGC T1-motni FW ATTCCGAUGGCTCCGAGGCTACTGGAGT T1-motni FW ACGCAAGUGCGACGTCTGATGCCAATAT T2-motni FW ATTCCGAUGCGGTTTTCAAGGATAACAGAT T2-motni RV ACGCAAGUCATGACCCCGGATAACTTTAAAA T3 motni FW ATTCCGAUAAGTCTTCCGTTACCCTTGCA T3-motni RV ACGCAAGUTGTAATTCCTACCTACCTACCTCTT Mammalian primers Primer name Sequence PGK FW ACGTCGCUCCGGTAGGCGCCAACCG PGK RV ATCGCACUGGCTGCAGGTCGAAAGGCC SV40 (+ori) FW ACGTCGCUCTGTGGAATGTGTGTCAGTTAGG SV40 (+ori) RV ATCGCACUAGCTTTTTGCAAAAGCCTAGG CMV FW ACGTCGCUCGATGTACGGGCCAGATATAC CMV-RV ATCGCACUATTTCGATAAGCCAGTAAGCAGT eGFP+K_GOI FW AGTGCGAUCGCCACCATGGTGAGCAA eGFP+K_GOI RV ATCGCTCUTTACTTGTACAGCTCGTCCATGC Mammalian backbone pU0020 FW ATTAAGCUAGTGAGTCGAATAAGGGCGACA Mammalian backbone pU0020 RV AGCGACGUGAGTCGAATAAGGGCGACACC eGFP_module FW AGTGCGAUCGCCACCATGGTGAGCAA eGFP_module RV ATCGGAAUTTACTTGTACAGCTCGTCCATGCC eGFP+K GOI FW AGTGCGAUCGCCACCATGGTGAGCAA eGFP+K GOI-stop RV ACCAGCGCUCTTGTACAGCTCGTCCATGCC eGFP_TS FW AGAGCGAUCGCCACCATGGTGAGCAA eGFP_TS RV ATCGGAAUTTACTTGTACAGCTCGTCCATGCC YFP_module FW AGTGCGAUCGCCACCATGGTGAGCAA YFP_module RV ATCGGAAUTTATCTAGATCCGGTGGATCCC YFP+K GOI FW AGTGCGAUCGCCACCATGGTGAGCAA YFP+K GOI RV ACCAGCGCU TCTAGATCCGGTGGATCCCG YFP_TS FW AGCGCTGGUCGCCACCATGGTGAGCAA YFP_TS RV ATCGGAAUTTATCTAGATCCGGTGGATCCC CFP_module FW AGTGCGAUCGCCACCATGGTGAGCAA CFP_module RV ATCGGAAUTTATCTAGATCCGGTGGATCCC CFP+K GOI FW AGTGCGAUCGCCACCATGGTGAGCAA CFP+K GOI-stop RV ACCAGCGCUTCTAGATCCGGTGGATCCCG CFP_TS FW AGCGCTGGUCGCCACCATGGTGAGCAA CFP_TS RV ATCGGAAUTTATCTAGATCCGGTGGATCCC mCherry module FW AGTGCGAUCGCCACCATGGTGAGCAA mCherry module RW ATCGGAAUCTACTTGTACAGCTCGTCCATGC mCherry+K GOI FW AGTGCGAUCGCCACCATGGTGAGCAA mCherry GOI-stop RV ACCAGCGCUCTTGTACAGCTCGTCCATGCC mCherry_TS FW AGCGCTGGUCGCCACCATGGTGAGCAA mCherry_TS RV ATCGGAAUCTACTTGTACAGCTCGTCCATGC BGH poly A FW ATTCCGAUCTGTGCCTTCTAGTTGCCAGC BGA poly A RV ACGCAAGUCCATAGAGCCCACCGCATC SV40 pA FW ATTCCGAUAACTTGTTTATTGCAGCTTATAATGGTTAC SV40 pA RV ACGCAAGUCAGACATGATAAGATACATTGATGAGTTTG Hygromycin FW ACTTGCGUCCAGCAGGCAGAAGTATGCA Hygromycin RV AGCTTAAUCAGGCTTTACACTTTATGCTTCC Neomycin FW ACTTGCGUCTGTGGAATGTGTGTCAGTTAGG Neomycin RV AGCTTAAUCAGACATGATAAGATACATTGATGAGTTTG Copenhagen primers Primer name Sequence pIPTG FW ACGTCGCUCAATACGCAAACCGCCTCTC pIPTG RV ATCGCACUTGTGTGAAATTGTTATCCGCTCA CYP79A1 FW AGTGCGAUaaagaggagaaaATGGCTCTGTTATTAGCAGTTTTT CYP79A1 RV ATCGGAAUTTAGATGGAGATGGACGGGTA CYP79B1 FW AGTGCGAUaaagaggagaaaATGTATTTACTTACAACGCTTCAAG CYP79B1 RV ATCGGAAUTACTTCACTGTAGGGTAAAGATGT 2C9 FW AGTGCGAUaaagaggagaaaATGGCTCGACAATCTTCTGGA 2C9 RV ATCGGAAUTTAATGGTGATGGTGATGGACAG Terminator FW ATTCCGAUCCAGGCATCAAATAAAACGAAA Terminator RV ACGCAAGUTATAAACGCAGAAAGGCCCAC Amp-cas FW AGCTTAAUTTACCAATGCTTAATCAGTGAGGC Amp-cas RV ACTTGCGUGACGTCAGGTGGCACTTTTCG Amplification of biobricks by PCR PCR MIX PCR mix 1 x PCR mix á 50µl 5 x HF PCR buffer with MgCl2 or GC buffer 10µl dNTP’s 2mM 5µl Primer forward 10 µM 4µl Primer reverse 10 µM 4µl Phusion DNA polymerase 5u/µl 0.3µl DNA template 0.5µl MilliQ water 26.20µl Procedure Start by mixing the receipt of PCR mix (cf. Materials) and remember to multiply by the amount of PCR reactions. Do not add DNA template to the PCR mix. Add the DNA template to the PCR tubes and add 50 µl PCR mix to each tube. After mixing the DNA template and PCR mix, the PCR mixture has to be run in the following PCR program. PCR Programs Temperature [°C] Time [min] Cycles 98 2:00 98 0:10 35 59 0:30 - 72 3:00 - 72 5:00 12 Store To check whether the PCR was succesful, gel electrophoresis was run on each PCR product. To check the PCR reactions were correct, all PCR samples were run on a Gel electrophoresis. All PCR reactions of 50µl were added 7-10µl loading buffer and run in 2% agarose. The Gel electrophoresis was set the volt to 75V and time after length but between 30-60 minutes. Purification of PCR Product Materials GFX PCR DNA and Gel band purification Kit from GE healthcare was used to purify the PCR product after gel electrophoresis. Preparations Wash buffer 1; Add ethanol to the buffer and mark the label. Store in airtight container. Procedures - GE healthcare protocol Sample Capture Weigh a DNase-free 1,5 ml microcentrifuge tube and record the weight. Using a clean scalpel, long wavelength (365 nm) ultraviolet light and minimal exposure time, cut out an agarose band containing the sample of interest. Place agarose gel band into a DNase-free 1,5 ml microcentrifuge tube. Weigh the microcentrifuge tube plus agarose band and calculate the weight of the agarose slice Add 10 μl Capture buffer type 3 for each 10 mg of gel slice, for example, add 300 μl Capture buffer type 3 to each 300 mg gel slice. Never use less than 300 μl Capture buffer type 3. Mix by inversion and incubate at 60°C for 15–30 minutes until the agarose is completely dissolved. Mix by inversion every 3 minutes For each purification that is to be performed, place one GFX MicroSpin column into one Collection tube. Sample Binding Centrifuge Capture buffer type 3-sample mix briefly to collect the liquid at the bottom of the DNase-free 1,5 ml microcentrifuge tube. Transfer up to 800 μl Capture buffer type 3- sample mix onto the assembled GFX MicroSpin column and Collection tube. Incubate at room temperature for 1 minute. Spin the assembled column and Collection tube at 16 000 × g for 30 seconds. Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin column back inside the Collection tube. Repeat Sample Binding steps b. to e. as necessary until all sample is loaded. Wash and Dry Add 500 μl Wash buffer type 1 to the GFX MicroSpin column. Spin the assembled column and Collection tube at 16 000 × g for 30 seconds. Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube (supplied by user). Elution Add 10–50 μl MilliQ water to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube. Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute. Spin the assembled column and sample Collection tube at 16 000 × g for 1 minute to recover the purified DNA. Proceed to downstream application. Store the purified DNA at -20°C. Gel electrophoresis Materials Agarose TAE Buffer (1L): 4.84 g Tris Base, 1.14 ml Glacial Acetic Acid, 2 ml 0.5M EDTA (pH 8.0), bring the total volume up to with water Sample Loading Buffer DNA ladder standard Electrophoresis chamber Gel casting tray and combs Procedures Measure 1.25 g Agarose powder and add it to a 500 ml flask Add 125 ml TAE Buffer to the flask. Melt the agarose in a microwave or hot water bath until the solution becomes clear. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. Place 20 µl of cybrsafe in 4-5 drops on the casting tray. Add agarose gel and mix until even. Let the gel cure for 30-45 min. Place the gel in a electrophoresis chamber. Make sure the TAE buffer in the chamber is 2-3mm above the agarose gel. On a piece of parafilm, mix 1 µl of PCR product with 3 µl of loading buffer. Load the gel with the samples, a negative, and DNA ladder. Run the gel at 70V. The time is depending on the bp length, but 30-60 min are usually good for 1000-3000bp. Visualise the gel in a -GEL EXPOSER!-. USER cloning Materials USER mix 1 x USER mix á 10µl USER enzyme 1 µl NEB (10 x diluted) 0,5 µl BSA 0,5 µl PCR product 8 µl Procedures Start mixing the USER mix (Table in materials.) Muliplying by the number of USER-clonings. PCR product must not be master USER mix. Transfer 2 µl of the USER mix to PCR tubes. Add the PCD products in an amount equal to 8µl for all components. Incubate for 37°C for 40 min and 30 min at 25°C. Tranformation in E.coli The preparations for the transformation can preferably be done while the USER cloning is incubating. Materials LB-plates with antibiotic resistance. ''E. coli DHα5'' cells" Digalski spartula Ethanol Bunsen burner Procedures Take LB- plates out of the refrigerator and mark them. Remember to use LB-plates with the right antibiotic. Take 50 µl competent ''E. coli DHα5'' cells per USER reaction, from the -80 freezer and place on ice. Additionally, place 1,5 ml tubes on ice. Add all USER reaction to the 50µl tube with the competent ''E. coli'' cells. Mix well by pipetting. Keep cells on ice for 30 min. Turn on the hot plate at 60°C. Heat chock each transformation for 90 sec. Afterward, put directly on ice for 2 min Plating of bacteria on LB-plates. Bacteria backbone with amp resistence gene. Sterilize a Drigalski spartula in 90% ethanol and flame between each transformation. Plate all the transformation mix out on the LB-amp plate and disperse with the cooled drigalski. Bacteria backbone without amp resistence gene -Add 500 µl LB to the transformation mix. -Incubate for 30-60 min at RT. -Spin the transformation mix down. -Remove the supernantant until approximately 50 µl are left. -Resuspend the pellet in the remaining LB. -Plate the 50 µl of tranformatin mix in the same way as described above, but on LB plate with the specific resistance gene. Incubate over night at 37°C. Next day; Check for visual colonies and use them for cultivating. Cultivation of transformed cells Material Liquid LB with resistance marker tooth pick Procedures Choose a couple of colonies from the LB-plate. Each colony are transferred to 5 ml LB + resistance marker with a pipet tip. Incubate over night at 37°C in the shaking incubator. Purification of plasmids Materials The GenElute Plasmid Miniprep Kit from Sigma-Aldrich was used to isolate our plasmids to use in fungi while QIAGENs EndoFree Plasmid Maxi kit was used to purify plasmids for use in mammalian cells. Preparation Thoroughly mix agents; Examine reagents for precipitation. If any reagent forms a precipitate, warm at 55–65 °C until the precipitate dissolves and allow to cool to room temperature before use. Resuspend Solution; Spin the tube of the RNase A Solution briefly to collect the solution in the bottom of the tube. Add 13 μl (for 10 prep package), 78 μl (for 70 prep package) or 500 μl (for 350 prep package) of the RNase A Solution to the Resuspension Solution prior to initial use. Store at 4 °C. Wash solution; Dilute with 95-100% ethanol prior to initial use. Procedures - Sigma-Aldrich protocol (use in fungi). Harvest cells: Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation. The optimal volume of culture to use depends upon the plasmid and culture density. For best yields, follow the instructions in the note below. Transfer the appropriate volume of the recombinant E. coli culture to a microcentrifuge tube and pellet cells at ;12,000 3 g for 1 minute. Discard the supernatant. Resuspend cells: Completely resuspend the bacterial pellet with 200 μl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous. Incomplete resuspension will result in poor recovery. Lyse cells: Lyse the resuspended cells by adding 200 μl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous. The lyse reaction must not exceed 5 min. Neutralize: Precipitate the cell debris by adding 350 μl of the Neutralization/Binding Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifuging at ≥12,000*3 g or maximum speed for 10 minutes. Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out of solution as a cloudy, viscous precipitate. Prepare columns: Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 μl of the Column Preparation Solution to each miniprep column and centrifuge at ≥12,000*3 g for 30 seconds to 1 minute. Discard the flow-through liquid. Load cleared lysate: Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at ≥12,000*3 g for 30 seconds to 1 minute. Discard the flow-through liquid. Wash column: Add 750 μl of the diluted Wash Solution to the column. Centrifuge at ≥12,000*3 g for 30 seconds to 1 minute. The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to remove excess ethanol. Elute DNA: Transfer the column to a fresh collection tube. Add 100 μl of MilliQ water to the column. Centrifuge at ≥12,000*3 g for 1 minute. The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C. Fungi Transformation in fungi Genetic Transformation of filamentous fungi – protocol from Center for Microbial Biotechnology (CMB) at DTU, Author Nielsen, J. B. Materials Media Minimal medium (MM) (1L): 50 mL D-glucose 20% w/V, 20 mL 50x mineral mix, 10 mL 1 M sodium nitrate, 20 g ager. Transformation media(TM)(1L): 342.3 g Sucrose, 20 mL 50x mineral mix, 20 g agar. Mineral Mix (1L): 26g KCL, 26g MgSO4·7H2O, 76g KH2PO4, 50 mL Trace element solution, MIlli-Q water to volume 1000 mL. D-glucose 20% (0.5 L): 100g D-glucose and MilliQ water up to 500 ml. Aspergillus protoplastationbuffer (APB): Final conc. 1.1 M MgSO4 and 10 mM Na-phosphate buffer. pH is adjusted with 2 N NaOH to 5.8. Aspergillus transformation buffer (ATB):Final conc: 1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2. PCT (200ml) - Final conc: 50% w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store at 4 °C. Procedures Initiation: The host strain is grown as three-point stabs on Minimal medium plates with the require suppliants added. MM will for convenience throughout the protocol refer to MM with the supplements included. Inoculation: The conidia are harvest by adding 5 ml of MM and firmly rub with a sterile Drigalsky spatula. The conidial suspension is pipette to a sterile 500 ml shake flask containing 100 ml MM. The cultures are incubated at 30 °C with 150 rpm of shaking over night (14-20 hours). Mycelial harvest: A funnel with a sterile Mira cloth (filter) is used to harvest mycelia. To remove residual glucose from mycelia the biomass are wash with Aspergillus protoplastationbuffer (APB). The filtered biomass is transferred to a new Falcon tube with a sterile spoon. Protoplastation: Mycellium is resuspenden in 10 ml filter-sterillized(0.45μm filters) APB containing 40 mg Glucanex/ml. The Glucanex is dissolved in APB with gentle magnetic stirring less than 100/min. Mycelia with dissolved Glucanex are mixed at 30 °C with 150 rpm of shaking for 2-3 hours. Portoplast solutions are diluted in APB adding up to 40 ml mark. An overlay of max. 5ml Aspergillus transformation buffer (ATB) diluted to ½x with sterile MilliQ-water is carefully placed on top of the APB. Centrifuged at 13 min 3000 RCF in Sorvall centrifuge. Protoplates should be observed as a halo of with slurry in the interphase of the two liquids. Withdraw of the protoplasts are done with pipette and placed in a Falcon tube. ATB is added up to 40 ml mark. Centrifuge at 300 RCF in 13 min and supernatanted are discarded. The protoplastes are resuspended in 1 ml ATB with a 5 ml pipette. Genetic transformation: Aliquotes of 50 μl are transferred to a 1.5 ml Eppendorf tube containgen 10 μl of DNA for transformation. Protoplast and DNA are incubated at room temperature for at least 30 min. Protoplat and DNA suspension are added to 1 ml PCT in a 15 ml tube and shake gently. Incubate for 1-5 min at room temperature. Dilute in 3 ml ATB. The tube is filled with molten transformation medium (TM) agar (temperature of 40-45°C) to apporeimately 12 ml. The tube is mix rapidly by inverting the tube twice. Poure directly on pre-made TM plates and incubate at 37°C for 3-8 days. Production of conidiospores Materials tooth stick LB plates Procedures Gently touch the a mature colony with a tooth pick. Hold the LB-plate upside down to avoid scattering of spores, and stab three different places. Incubated plates at 37°C for approximately five days. After incubation spores were either harvested from the plates or they were stored in the fridge at 4°C. Flourescence Microscopy Materials MM Bleomycin 4-NQO Procedure Cultivation for fluorescence detection was performed by collecting the conidia from plates having three point stab colonies that have grown for 4-5 days. 5ml of MM with supplements were added to the plate and conidia were rubbed off the colonies with a sterile Drigalsky spatula. Inoculate the conidial suspension in 5ml MM in a sterile 50ml Falcon. Add bleomycin (10mg/mL) and 4-NQO (5 mg/mL). This will cause damage to the DNA inducing gene expression Incubated at 37 °C with 150 rpm of shaking overnight. Restriction enzyme analysis Materials Ingredients Volume: 10µl Device 5 µl Buffer 3 3 µl BgI II 1,5 µl H2O 5,5 µl Procedures Mix the recipe for restriction analysis of the fungi plasmid from the table in materials. Incubate for 1 hour Run gel electrophoresis Fluorescence detection Materials Coverslide + overlay cover. Conidia Culture. Procedure Take 500µL of conidia culture (ref. till protocol for det) in a tube. This has to be done after 4, 24, 48 and 120 hours . Centrifuge the tube at 16000g for 1min and 30 sec. Discard the supernatant. Resolute the pellet in 5µL Milli Q water and place on slide. Place the overlay cover Detection microscopy was performed first with DIT filter and fluorescence filter with enlargement of 10x and 100x. To check background fluorescence, comparison of detection with yellow filter was made against detection by green and red filter. Extraction of proteins Materials Z-buffer AEBSF stock solution Bradford Reagent BSA MilliQ water Minimal media with aminoacids Procedure Inoculation of conidia in shaking bottle The colonies from the three point stab are harvested by adding 2mL MilliQ water to the petri dish. The colonies are rubt with a digalski spartula. 500µl of the colony suspension is pipette into a 500mL shaking bottle with 100mL minimal media containing amino acids Incubate in 48 hours at 37°C Protein extract 2 mL culture are transferred from the shaking bottles to eppendorf tubes. Remember to do triple determination. Centrifuge tubes in 1 min with 8000g Remove the supernatant. Transfer the mycelierne with a sterile spartula to FastPrep tubes. 250 µL Z-buffer is added to the FastPrep tubes in stink cabinet. Add approximately 200µL of the small glass balls to the FastPrep tubes. Add 12,5µL AEBSF stock solution. Place the FastPrep tubes in the FastPrep machines. Let it run with max velocity for 30 sec. Tubes are kept on ice from now on. Add 250 µL Z-buffer and mix well. The extract is transferred by a 1000 µL pipette. Place the pipet at the bottom of the tube and transfer it to a new eppendorf tube. Centrifuge the eppendorp tubes in 15 min at 10000g to purify the protein extract. Transfer the supernatant to a new eppendorp tube. Assays Materials Z-buffer AEBSF stock solution Bradford Reagent BSA MilliQ water Minimal media with aminoacids Protein extract (from above) ß-galactosidase assay Add 25 µL extract and 225 µL Z-buffer to a well in a microtiter plate. Three wells per extract for triple determination. Mix enough ONPG stock solution so 50 µL can be added to each well. Measure OD with the computer program Gen5; Shake the plate every 30 sec to mix the solution, rest for 10 min before measuring the OD. Bradford assay Add 5 µL extract and 245 µL Bradford reagent to a well in a microtiter plate. Three wells per extract for triple determination. Make a BSA standard with concentrations between 0,1-2,0mg/mL with. See table below: Preparation of BSA standards Standard µL standard µL dH2O Concentration Std 1 300 µL stock 1200µL 2 mg/ml Std 2 200 µL stock 1000µL 1,67mg/ml Std 3 700 µL Std 1 700µL 1 mg/ml Std 4 700 µL Std 2 700µL mg/ml Std 5 700 µL Std 3 700µL 0,5 mg/ml Std 6 700 µL Std 4 700µL mg/ml Std 7 700 µL Std 5 700µL 0.25mg/ml Std 8 700 µL Std 7 700µL 0,125mg/ml Std 9 - 700µL 0 mg/ml Make triple determination. Incubate at 37°C for 10 min. Mammalian cells Cell culture and reagents Materials cells: U2OS cells were kindly provided by The Danish Cancer Society. U2OS cell line is derived from the bone tissue of a patient suffering from osteosarcoma. U2OS cells show epithelial adherent morphology. Medium: 500 ml DMEM 50 ml Fetal Calf Serum (FCS) 5 ml Penicillin 5 ml Streptomycin Procedures Add FCS, penicillin, and streptomycin to a new flask of DMEM. Store at 5°C. The cells are cultivated in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Penicillin, Streptomycin, and 10 % heat-inactivated foetal calf serum (FCS). Penicillin and Streptomycin are added to prevent any microbial growth. FCS is added to supply essential non-defined components, such as serum proteins and lipids. Supplemented DMEM medium is referred to as complete DMEM throughout the report. The cells are adherent and are kept in 75 cm2 culture flask until 80-100% confluency, where they are passed on to new culture flasks or cover slips. Cultivation of cells Materials 25 cm2 culture flask Complete DMEM Procedures Place the appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells (about 1-2 hours before). The U2OS cells are kept at -80°C until defrosted gently at 37°. Exactly when defrosted, they are mixed with 12 ml complete DMEM in the pipette. The cell suspension is transferred to a 15 ml vial and centrifuged at 280 g for 10 minutes. The supernatant is discarded. The cell pellet is resuspended in 5 ml complete DMEM and transferred to a 25 cm2 culture flask. The cells are kept at 37°C in a 5% CO2 incubator until the following day, where they are passed on to larger culture flasks. Passing and maintenance of U2OS cells Materials 75 cm2 culture flask: 50 ml vial Complete DMEM medium 0.05 % EDTA-trypsin Procedure The appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells. The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use. 1 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly. 1 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2. 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them. 2,5 ml (depends on the concentration of the cells) cell suspension is transferred to a new 75 cm2 culture flask. The cells are kept at 37°C in a 5% CO2 incubator until they are 80-100% confluent. This takes about 2-3 days. Then they are passed on to a new flask or plate. Transferring the cells to coverslips Materials 75 cm2 culture flask: 50 ml vial Complete DMEM medium 0.05 % EDTA-trypsin 6-well plate Cover slips Procedure The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use. 1,5 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly. 1,5 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2. 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them. Calculate the concentration of cells you want in the wells Cover slips are placed in the bottom of a 6-well plate (2 cover slips/well). 2 ml cell suspension is added gently to each well. The plate is placed in the incubator at 37°C and 5% CO2 O/N. Transfection of cells Materials Optimem Fungene 6 Plasmid DNA Procedure Disinfect LAF bench and gloves with ethanol before and after working in the LAF bench. To prepare the transfection, transfer 46μl optimem to a 1,5ml eppendorf tube per tranfection. Add 3 μl of Fungene 6 directly into the optimem and pipet up and down to mix. Flick gently on the tube to obtain further mixing Incubate for 5 mn at room temperature Add 1 μl of plasmid DNA directly into the optimem/Fugene mixture and pipet up and down to mix. Flick gently on the tube to obtain further mixing. If there is any liquid remaining on the side of the tube, spin very shortly in the centrifuge. Incubate the eppendorf tubes for 15 min at 37°C. Take out the 6 cm2 dish with HEK293 cells from the incubator. In the LAF bench, add the 50 μl transfection mixture to the cells. Do it drop-wise into the medium. Use rocking motion to gently mix and place the dishes back in the incubator. Coverslides for microscopy Materialer PBS MilliQ water Formaldehyde lint-free paper Cover slides Transparent nail varnish Procedure Delute PBS with MilliQ ten times, having final amount of 500ml. Sterile move the coverslips to a 24-well plate. Wash twice with PBS. Add 400 μl Formaldehyde to each well under the fume hood. Incubate for 12 min at RT. Remove liquid to the waste bin Wash three times with PBS. Remove coverslips and immerse shortly in MilliQ water before laid on lint-free paper for drying. Take 4 μ Vecta shield and place on cover slide. Repeat for each coverslip. When the coverslip is dry, place on coverslide with cells downwards. Fixate the coverslips with transparent nail varnish. Purification of plasmids Mateials EndoFree Plasmid Maxi kit from QIAGEN was used to purify plasmids for use in mammalian cells. Procedures - QIAGEN protocol Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for ~8 h at 37°C with vigorous shaking (~300 rpm). Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 100 ml medium, and for low-copy plasmids, inoculate 250 ml medium. Grow at 37°C for 12–16 h with vigorous shaking (~300 rpm). Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. Resuspend the bacterial pellet in 10 ml Buffer P1 Add 10 ml Buffer P2, mix gently but thoroughly by inverting 4–6 times, and incubate at room temperature for 5 min. The lysis reaction time must not exceed 5 min. Add 10 ml chilled Buffer P3 to the lysate, and mix immediately but gently by inverting 4–6 times. Proceed directly to next step. Do not incubate the lysate on ice. Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for 10 min. Do not insert the plunger! Remove the cap from the QIAfilter Cartridge outlet nozzle. Gently insert the plunger into the QIAfilter Maxi Cartridge and filter the cell lysate into a 50 ml tube. Add 2.5 ml Buffer ER to the filtered lysate, mix by inverting the tube approximately 10 times, and incubate on ice for 30 min. Equilibrate a QIAGEN-tip 500 by applying 10 ml Buffer QBT, and allow the column to empty by gravity flow. Apply the filtered lysate from step 9 to the QIAGEN-tip and allow it to enter the resin by gravity flow. Wash the QIAGEN-tip with 2 x 30 ml Buffer QC. Elute DNA with 15 ml Buffer QN. Precipitate DNA by adding 10.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant. Wash DNA pellet with 5 ml of endotoxin-free room-temperature 70% ethanol (add 40 ml of 96–100% ethanol to the endotoxin-free water supplied with the kit) and centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of endotoxin-free Buffer TE. "To determine the yield, DNA concentration should be determined by both UV spectrophotometry and quantitative analysis on an agarose gel."