Protocols
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201 | UT | 201 | cloning | |
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UTSW_standard purification | UTSW | UTSW_standard purification | purification | Purification Cell disruption: 1. Spin 1L bacterial cultures at 4000rpm 40mins at 4 ºC. 2. Prepare BB buffer. 3. Decant the media (incubate with bleach for at least 1hour before you pour it into sink) 4. Resuspend the cell plate in cold BB buffer (35ml of BB per 1L of bact. Culture), transfer the suspension into 50mL plastic tubes, label and freeze at -80 ºC. 5. Thaw cells in cold water and protease inhibitors (1mM final concentration of PMSF and benzamidine) 6. Sonicate on ice for 5X30s with a 30s rest in between. Repeat if the lysate is still viscous. 7. Take 30 µL sample for SDS-PAGE, spin 20mins (microfuge) at 13000rpm, separate soluble fraction from sediment, label SUP and SED and add 15 µL of loading buffer with DTT and heat at 99 ºC for 3mins. 8. Freeze the lysate at -80 ºC. Buffer Preparation: Prepare buffers for purification, check pH and filter them. BB buffer: 500mM NaCl (50ml/500ml of 5M NaCl) 5% glycerol (25ml/500ml of 100% glycerol) 50mM HEPES pH 7.5 (25ml/500ml of 1M HEPES pH 7.5) 5mM imidazole (2.5ml/500ml of 1M imidazole) WB buffer: 500mM NaCl (50ml/500ml of 5M NaCl) 5% glycerol (25ml/500ml of 100% glycerol) 50mM HEPES pH 7.5 (25ml/500ml of 1M HEPES pH 7.5) 30mM imidazole (15ml/500ml of 1M imidazole) EB buffer: 500mM NaCl (50ml/500ml of 5M NaCl) 5% glycerol (25ml/500ml of 100% glycerol) 50mM HEPES pH 7.5 (25ml/500ml of 1M HEPES pH 7.5) 250mM imidazole (175ml/500ml of 1M imidazole) 5mM β-mercaptoethanol must be added to all buffers before use (117 µL/500m of 100% β-mercaptoethanol) Dialysis Buffer # 1 500mM NaCl (400ml/4L 5M NaCl) 5% glycerol (200ml/4L 100% glycerol) 10mM HEPES pH 7.5 (80ml/4L 0.5M HEPES pH 7.5) Same buffer for Dialysis Buffer # 2(1L) and Gel filtration buffer(2L) 200mM NaCl (120ml/3L 5M NaCl) 100mM HEPES pH 7.5 (600ml/3L 0.5M HEPES pH 7.5) 5% glycerol (150ml/3L 100% glycerol) First affinity chromatography: 1. Spin the lysate at 12000 rpm at 4 ºC for 40min and filter it through 0.8µm filters. 2. Prepare columns as follows: - DE52 (Whatman): mix 10gms of DE52 in 100ml of 2.5M NaCl, add to large column and equilibrate with 40ml of BB buffer - Ni resin (Quiagen): add 5ml bed volume to small column and equilibrate with 20ml BB buffer. - Place column in series (DE52 → Ni resin) 3. Save the sediment after centrifugation at -80ºC (can be removed after the purification is over and OK) 4. Apply the clarified lysate to upper DE52 column and let it flow through both columns, collect flow-through fraction (FT) 5. Take 30 µl sample for SDS-PAGE, label FT and add 15 µl of loading buffer with DTT and heat at 99ºC for 3min. 6. Add 2x10 ml of BB buffer to DE52 column ( don’t collect the flow-through fraction) 7. Remove the DE52 column and work further only with Ni column. 8. Wash Ni column with 40ml WB buffer and collect WB fraction. 9. Take 30µl sample for SDS-PAGE, label WB and add 15µl of loading buffer with DTT. 10. On the ‘last drop’ of WB perform Bradford assay to determine if all protein were washed out. If not wash with WB buffer further until OD595nm is ~ 0.05. Don’t collect fraction. 11. Let the column dry briefly, add 2ml of EB buffer and let sit for 1min. Elute the protein sequentially with 10ml of EB buffer while checking protein content by Bradford assay. Stop elution when OD595nm is ~ 0.05. Collect all elution into one 50ml tube, label EB (ideally the volume should not exceed 50ml) 12. Take 30µl sample for SDS PAGE, label EB and add 15µl of loading buffer with DTT and heat at 99ºC for 3min. 13. Add EDTA to EB fraction to final concentration 1mM. 14. Add TEV protease to EB fraction(follow the instructions on box with TEV prep) 15. Dialyze O/N into 4L of Dialysis buffer #1 at RT. Do not add more than 80ml of sample into 4L dialysis buffer. Second affinity chromatography: 1. Take 30µl sample for SDS PAGE from dialysis bag, label TEV and add 15µl of loading buffer with DTT and heat at 99ºC for 3min. 2. Run SDS-PAGE gel with samples from purification and after TEV cleavage. 3. If gel shows that His-Tag is cleaved out you can proceed to second affinity chromatography step. If cleavage was not sufficient, allow the protease digest further. 4. Add protease inhibitors to the content of dialysis tube (1mM final concentration of PMSF and benzamidine). 5. Add 6ml of Ni resin pre-equilibrated in BB buffer and shake at 4ºC for 40mins. 6. Pour the suspension into small column and collect the flow through (label FT2) 7. Then wash the column by 5ml of BB several times until all protein is washed out (as determined by Bradford assay), collect as BB2. 8. Take 30µl sample for SDS PAGE from FT2 and BB2, add 15µl of loading buffer with DTT and heat at 99 ºC for 3mins. 9. Dialyze FT2+BB2 O/N into 4L of dialysis buffer #2 at 4ºC. Do not add more than 80ml of sample into 4L of dialysis buffer. Gel filtration: Setup HPLC column – Superdex 75 HR 10/30. Collect elutes and run SDS-PEGE and Protein concentration: 1. Run SDS PAGE gel with samples from purification and after TEV cleavage. 2. While running gel, start protein concentration by ultra filtration using Vivaspin concentrators. Concentrate protein to ~50mg/ml or until precipitation occurs, remove from concentrator and determine volume. Wash concentrator with equal volume of 10mM HEPES pH 7.5 and pool the 2 fractions. 3. Spin at 13000rpm and determine concentration by OD280nm. 4. Take 10µl sample for SDS PAGE add 5µl of loading buffer with DTT and heat at 99 ºC for 3mins. 5. Take 10µl sample for SDS PAGE add 5µl of loading buffer without DTT and heat at 99 ºC for 3mins. 6. Take 10µl sample for native PAGE add 5µl of native loading buffer. 7. Aliquot the protein, label and store at -8 ºC Protein analysis before crystallization: Run SDS PAGE gel and native electrophoresis gel. |
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NU_default_purification | NU | NU_tag-on_purification | purification | Proteins are purified by Ni-affinity chromatography on HiTrep FF and desalting HiPrep 20/10 columns using AKTA Express system. Pure proteins are collected in buffer, containing 10 mM Tris-HCl (pH 8.3, 500 mM NaCl and 5 mM b-mercaptoethanol. Samples are concentrated to 5-25 mg/ml depending on solubility and used for crystallization experiments. Unused protein is flash-frozen in liquid nitrogen and stored at -80C. |
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standard purification_ANL | ANL | standard purification_ANL | purification | Day 1 – Small Culture (50mL) Pick up cell culture from cloning lab. They will be in small, 2mL test tubes. Each tube contains a different target protein cloned in pMCSG7 (or other MCSG) vector and expressed in E.coli BL21 (DE3) cells. 1. Prepare M9 “Pink” Media by adding the following in a 1L bottle: • 1 pouch M9 Salts + NIAAC • 10 mL Metal Supplement solution • 1 mL Kan/Amp/B1/B12 solution • 10 mL of 50% glycerol • 980 mL MQ water 2. Aliquot 50 mL of M9 “Pink” Media in small, 250mL-plastic bottle 3. Add 100 uL of LB cell culture 4. Shake in incubator overnight, 37 ºC, 150 rpm Day 2 – Large Culture (1 L) 1. Prepare M9 “Pink Media” as shown above, except add 930 mL of MQ water 2. Add 25 mL of small culture from small plastic bottle to each 1 L bottle. Place an absorbent underpad on the cart prior to the transfer of bacterial cultures (e.g. Fisher # 14-206-63)). See special section at the end of this document for handling spills for this step. 3. Shake in incubator at 37 ºC, 200 rpm until cells grow to OD 1.0 -1.4. You should start measuring OD (595 nm) after about 2.5-3 hours. 4. Add 30 mL of IAAC + Se-Met solution 5. Transfer bottles to cooled 4 ºC incubator and shake for 1 hour 6. Increase incubator temp to 18 ºC; induce the cells by adding 0.5 mL of 1 M IPTG 7. Grow cells at 18 ºC overnight Day 3 – Harvesting Cells 1. Pour cell culture in 1L centrifuge bottle. Centrifuge for 8 minutes @ 7000 rpm, SS-6000 rotor (use the cart covered with an absorbent pad for liquid handling and see special section at the end of this document for handling spills) 2. Dispose supernatant into 1:10 diluted bleach solution. Pour into sink after 20 minute incubation. 3. Keep cells in 4 ºC from now on 4. add 1 tablet of Protease Inhibitor 5. Re-suspend the cells by adding 25 mL of cold Lysis Buffer and gentle shaking 6. Transfer resuspended cells into 50 mL Falcon tube and bring volume up to 40 mL with lysis buffer 7. Freeze cells in -80 ºC freezer until they are ready to be purified Day 4 – IMAC 1* *Note: AKTA must be stripped, charged, and blanked before actual purification run 1. Thaw out cells - place in bucket of warm water for a few minutes. When thawed, transfer to ice (4 ºC ) 2. prepare Lysozyme stock solution: 50 mg/mL. Add Lysozyme to cells @ final concentration 1mg/mL 3. Mix gently and keep cells on ice for 30 - 60 min 4. Sonicate cells on ice for 3 minutes. Program: 4 seconds “on” 12 seconds “off”. Wear ear muffs during the procedure. After the sonication process is complete wait 5 minutes before opening the sonicator housing. Wipe the sonicator tip with gauze (wet the gauze with 10% bleach, allow 20 minutes of contact time), rinse the tip with distilled water and wipe. Discard the gauze in an autoclave bag. 5. Transfer to 45 mL centrifuge tubes. Centrifuge for 1 hour @ 17,355 rpm, SS-34 rotor. Place an absorbent pad on the benchtop or cart during liquid transfer. 6. Using 0.4 μm syringe filter, filter supernatant into clean, 50 mL Falcon tube 7. Run Purification Program in AKTA Xpress Buffers for AKTA Xpress Lysis Buffer Elution Buffer Desalting Buffer HEPES pH 8.0 50 mM 50 mM 50 mM NaCl 500 mM 500 mM 500 mM Glycerol 5 % 5 % 5 % *β-mercaptoethanol 10 mM 10 mM 10 mM *Imidazole 20 mM 250 mM none *4X Buffer A does not contain these ingredients To Prepare Buffers – add ingredients, stir, filter and store at 4 ºC Stock Solution Lysis Buffer (4L) Elution Buffer (2L) Desalting Buffer (4L) 4X Buffer A 1 L 500 mL 1 L 14.3 M β-mercaptoethanol 2.8 mL 1.4 mL 2.8 μL 2.5 M Imidazole 32 mL 200 mL None MQ Water Bring up to proper volume Other Stock solutions needed for AKTA Xpress: 50 mM EDTA/0.5 M NaCl 100 mM NiSO4 Please use the laminated sheet and indicate the protein identifier(s) and your name for those targets of unknown function of RG2 microbes. The sheet must be attached to the chromatography refrigerator where the purifcation procedure takes place. Day 5 – TEV Cleavage 1. Collect and combine fractions, keep everything at 4 ºC 2. Take 50 uL of each pure protein sample. This will be the “uncut” samples – proteins that still contain His tags. 3. Calculate amount of TEV protease needed 4. Add TEV to remainder of protein sample 5. Incubate proteins at 4 ºC refrigerator for 1-3 days 6. Run SDS-PAGE to determine if tag cleavage has been successful. Use “uncut” protein samples as a reference point. 7. If successful, run IMAC 2 Day ? – IMAC 2 1. Add 4 mL of Ni-NTA slurry to a plastic disposable column 2. Allow column to “pack” by letting liquid flow through 3. Wash packed column with 20 mL MQ water 4. Equilibrate with Lysis Buffer 5. Slowly add protein sample using transfer pipette 6. Collect flow-through sample right away 7. Measure volume and concentration of protein |
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standard cloning_ANL | ANL | standard cloning_ANL | cloning | Day 1: Setup PCR plate, and run it in the thermocycler overnight. This protocol will produce a well volume of 51 µL: 0.26 mM each primer 1x buffer 0.2 mM each dNTP 1 mM MgSO4 0.016 U/µL KOD polymerase Notes: - Different gDNAs will be used in different wells, as specified in the plate design. - gDNA is added in the amount of 8.333 ng/well. Stock gDNA should be 100 ng/µL. - KOD polymerase should not be thawed. It is a glycerol solution, and you can pipet it at -20°C. - KOD polymerase is HotStart; inactive until heated. Not necessary to add the polymerase last. Hazards: Normal Precautions. 1. Begin thawing reagents: 10x KOD buffer dNTPs MgSO4 gDNA primer plates 2. Prepare a master mix according to the PCR Setup spreadsheet. Keep on ice at all times. 3. Divide the master mix into different segments for the different gDNAs, as specified in the spreadsheet. 4. Dispense 40 µL of the correct PCR reaction mix into each well in the PCR plate. 5. Keep PCR plate on ice, and run program “primersetup” to add primers. 6. Centrifuge plate briefly to ensure all liquid is at the bottom of the wells. 7. Run program KOD1 in thermocycler. Denature 95°C for 3 minutes Cycle(32x) 95°C for 40 seconds 53°C for 40 seconds 72°C for 1.5 minutes (depends on anticipated product length) Polish 72°C for 5-10 minutes Store 4°C overnight. Runtime: ~2 hours and 30 minutes Day 2: Verify PCR with Picogreen assay. Purify PCR products. Hazards: “No data are available addressing the mutagenicity or toxicity of Quant-iT PicoGreen dsDNA reagent. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.” – PicoGreen manual. - Try to avoid any contamination of PicoGreen, clean up spills immediately, change gloves if any spills occur. Change gloves after procedure. Notes: PicoGreen has a high freezing point, and takes a while to thaw. The data from FluoroStar is delivered by rows, while SG data is usually kept in columns. The data must be re-sorted before pasting into your SG### spreadsheet. 1. At least 20 minutes prior to setup, take the Picogreen dye out of the fridge and allow it to thaw at room temperature, in a dark location. 2. Assemble materials. PCR frags plate TE buffer, 25 mL Black flat bottom 96 well plate Reservoir with water Reservoir for diluted PicoGreen 3. Dilute PicoGreen reagent. 25mL of TE are mixed with 125 μl of picogreen dye for every two plates processed. Be sure to use this mixture immediately. It is light sensitive; therefore the longer it sits out in the light, the more your results will be affected. 4. Run Multimek program PicoG1. Setup as below: 5. 6. Setup Fluorostar software. Open program. Highlight “User” and click “Run”. Set mode to “Fluorescence”. (box in bottom left of window) 7. Open the top of the FluoroStar machine and rotate the cords so the Fluorescence cords (silver cords) are at the top. 8. Open the Fluoro tray (button at top of window). Place the fluorescent plate. Close the Fluoro tray. 9. Start the program. Click on the “Traffic Signal” icon. Select the “Picogreen Assay” program, and press “Ok”. Name your experiment run. Click “Start Test Run”. 10. Your results are put into an Excel spreadsheet. Access this spreadsheet by clicking the “Microsoft Excel” icon in the toolbar. In the spreadsheet, select your test run and double click. Overview: Remove primers and dNTPs from the PCR products. End result is your gDNA and amplified genes, in 150 µL of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) or EB buffer (10 mM Tris·Cl, pH 8.5). Be careful if eluting with TE, the EDTA may inhibit subsequent reactions. Hazards: Robot; keep clear. Notes: Watch the robot to make sure that all vacuum steps are performed as expected. It is especially important that the PE buffer is all pulled through before you add EB / TE. Materials: PM, PE, and TE buffers QiaQuick (purple) filter Deepwell plate Skirted PCR plate 1. Open the Biomek software, and open program “PCR Purification”. Setup the table as indicated in program. a. Put your PCR plate in the drilled bottom plate. b. Be sure to fill the PE reservoir to the brim, the program uses a lot. 2. Run the program. Be sure to turn on vacuum and vacuum switch as instructed, and to blot when instructed. If you have filled the PE reservoir to the brim, there is no need for the mid-procedure refill. 3. Be sure to turn the vacuum pump off. Day 3: Make your PCR fragments LIC-ready. Perform LIC and transform into BL-21 cells. LIC Overview: Use the 3’ to 5’ exonuclease activity of T4 polymerase to create overhangs on your PCR frags. Combine 32 μL of PCR pur with 10.8 μL of LIC rxn mix. Final concentrations of LIC reaction in plate: 0.94x T4 polymerase buffer 2.21mM dCTP 4.34mM DTT 2 units T4 DNA polymerase (0.05U/μL) Notes: This procedure is modified. No mineral oil, no PCR pur dilution. Hazards: Robot; keep hands clear. DTT is an irritant and health hazard; avoid contact. Materials: 2 skirted 96 well plates LIC reaction materials 8. Make LIC Reaction mix as follows for two plates: 1000μL 10x T4 Polymerase buffer 232.5μL 100mM dCTP 45.6μL 1M DTT 1160μL Water 200μL 2.5U/μl T4 DNA Polymerase (Novagen-LIC quality) 2638μL Total 9. Array 10.8 μl of LIC reaction mix into the labeled MJ Thermocycler plates. 10. Multimek – Run program LICxGC. This will transfer 32 µL of PCR pur to the LIC plate, and pipet mix. 11. 12. Foil seal the plates. Incubate at RT for 30 min (up to an hour is permissible). 13. Inactivate enzyme by incubating at 75°C for 20 min. 14. Store at 4°C short term, -20°C for up to several months, or -80°C for even longer periods. Overview: Anneal the prepared insert into the prepared vector. Then transform and plate. Hazards: LIC insert solution contains DTT. Materials: 48-well LB/amp plates Competent cells, 5 mL / plate (10 mL for 2 plates) Vector 1. Begin gently thawing comp cells on ice. Label new plate for annealing. 2. Array vector into annealing plate. 3. Add LIC prepared insert, 4 µL per well. 4. Incubate at RT for 10 minutes (5 minutes to 1 hour is ok). 5. Incubate on ice for 2 minutes. 6. Add competent cells, 55 µL per well. 7. Incubate on ice for 5 minutes, to allow cells to recover. 8. Heat shock at 48°C for 45 seconds, to make cells receptive. 9. Incubate on ice for 2 minutes. 10. Add LB medium (SOC is preferable), 60 µL per well (or up to 120 µL) 11. Incubate at 37°C for 30 minutes (up to 1 hour is ok). While they are incubating, prewarm the agar plates. 12. Plate 60 µL. Leave plates uncovered to dry, then invert and incubate at 37°C for ~14 hr. Plates must be dried to separate individual colonies. |
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standard expression_ANL | ANL | standard expression_ANL | expression | Overview: Grow cultures, induce protein expression. Materials: M9 components. Deepwell plates Thermocycler plates Notes: The red solution includes 100 mg/mL amp. Day 1: Starter Culture. Start procedure in the afternoon. 1. Warm growth plates. Thaw amp. 2. Add ampicillin 100ug/mL to LB. Array LB broth 1 mL/well into your deepwell plate. 3. Using a multichannel pipet tip, select a single colony from each agar plate square, and inoculate the appropriate well the growth plate. Leave tip in. Repeat on a new plate, so you have an A and B clone. 4. Leave tips in, and cover with breathable sealing tape. Incubate overnight 37°C, shaking. Day 2: Induction 1. Noon: make M9 media. 979 mL water 10 mL mineral supplement solution 10 mL 50% glycerol solution 1 mL Kan + Amp + vitamin solution 1 pouch of M9 salts 1 L total 2. Array 1mL/well into a deepwell plate. This is your induction plate. 3. Inoculate the induction plate with 40µL of your starter culture. Centrifuge remaining starter culture at 1700rcf for 15 minutes. Dump supernatant, freeze pellets for later plasmid purification. 4. Incubate at 37°C, shaking, for 4 hours until the OD600 is 0.8 -1. 5. Turn temperature down to 4°C, incubate for 30 min. 6. Mix 40µL of induction plate with 40µL of 50% glycerol. Freeze this glycerol stock. 7. Add 30µL of IAAC + Sel-Met + IPTG solution. Grow overnight at 18C. Day 3: Harvest & Lysis 1. Centrifuge your induction plate at 1700rcf for 15 minutes; discard supernatant and freeze pellets. 2. Prepare lysis buffer: 15 mL of 4X lysis buffer 45 mL of water 300 µL of 1M DTT 1 tablet of Roche protease inhibitor Mix thoroughly, then add: 20 µL of rLysozyme and Benzonase 3. Add 125 µL of lysis buffer to each well of your frozen cell pellets. 4. Vortex thoroughly, until all pellets are resuspended. 5. Incubate at 25C, shaking, for 30 min. 6. Freeze on dry ice, 15 min. 7. Sonicate frozen plates for 1.5 min each, at around 250W. Keep water bath as cold as possible. 8. Freeze-sonicate plates (as in steps 4 and 5) twice more. 9. Incubate at 25C, shaking, for 30 min. 10. Freeze-sonicate plates (as in steps 4 and 5) three times. 11. Remove 10uL of sonication mixture, and mix with 30 µL of SDS loading buffer. Boil and freeze. This is your expression sample. 12. Prepare TEV digestion mix: 0.5 mL 4X lysis buffer 1.5 mL water 10 µL 1M DTT 200 µL 0.5M EDTA 3 mL TEV crude extract 13. Add 10uL of TEV digestion mix to 90 µL of sonication mixture in a new conical plate. 14. Spin down at 1700rcf, (do not dump supernatant though) leave at 4C overnight. Day 4. 1. Spin down at 1700rcf. Remove 40 µL of the supernatant (make sure not to get any of the pellet). Add to 20 µL of 4X SDS loading buffer. Boil and freeze. This is your solubility sample. Day 5. Overview: Purify plasmids from expressing clones, for storage and future transformation. Hazards: Normal precautions. Procedure includes alkaline, acidic, combustible, and irritant solutions. Materials: - Frozen pellets from starter cultures. - New deepwell plates. - Buffers: P1, P2, N3, PE, TE (or EB). - Turbo Filter and QiaPrep Filter. - New skirted plates. Notes: Use cone shape deepwell plates only. 1. Resuspend frozen pellets in 150 µL of Buffer P1. Vortex thoroughly. 2. Use Biomex FX to combine the best-expressing clones into one plate. 3. Add 150 µL of Buffer P2. Shake gently, do not vortex. Add N3 after 5 minutes. This timing is important here… try to get it right around 5 minutes. 4. Add 250 µL of Buffer N3. Shake gently, do not vortex. 5. Incubate on ice for ~20 minutes, to allow greater precipitation of ssDNA, KDS, etc. 6. Proceed with FX program “Plasmid Purification” |
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NU_default_crystallization | NU | NU_robot_crystallization | crystallization | Crystallization experiments are usually performed by vapor diffusion method using Phoenix RE crystallization robot with 96-DWBlock screens from Qiagen and sitting drop 96-well crystallization plates. Every reservoir well contains 100ul of screen solution and every sample well- 1ul of protein (concentrations 7-10mg/ml) mixed with 1ul of reservoir solution. |
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Cloned by collaborators | Cloned by collaborators | cloning | Cloned by collaborators | |
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Target_selection | UCL | Target_selection | selection | |
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NU_default_cloning | NU | NU_default_cloning | cloning | Most of the cloning is done at JCVI according to their protocol. Some of the community targets are cloned in the requester laboratory. |
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4 | UT | 4 | cloning | pET28b |
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CSG-003_PCR Amplification and Ligation Independent Cloning (LIC) | JCVI | JCVI_LIC_protocol | cloning | PCR Amplification and Ligation Independent Cloning (LIC) as described in detail in the SOP CSG-003: see CSG-003_PCR_and_LIC_v002.pdf |
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Northwestern HSQC protocol | Northwestern HSQC protocol | nmr | Standard protocol for HSQC used at Northwestern University | |
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18 | UT | 18 | cloning | Escherichia coli DH5a |
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17 | UT | 17 | cloning | pMCSG7 |
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uva_expression_semet_1 | UVA | UVA Se-Met expression protocol | expression | 1. Start cultures from frozen stocks of expression strain of E.coli. BL21(DE3)RIPL. 2. Plate them on an selective antibiotic plate. 3. Incubate plate overnight at 37 degrees C. Day 2 4. To start pre-culture, pick a single colony and transfer into 20 mL of M9 Minimal media (by Orion Enterprise Inc) with selective antibiotic and grown at 37 degrees C. Day3 5. Prepare M9 Minimal Media according to the manufacturer protocol enclosed in the package. 6. Add 20 mL of pre-culture to 950 mL of M9 Minimal Media with selective antibiotic. 7. Grow at 37 degrees C until OD600 reaches 0.8-1.6. 8. Collect a sample of non-induced bacteria at this point. 9. Lower the temperature to 16-20 degrees C. 10. Add 20 ml of SeMet, wait 15 min. 11. Add IPTG to a final concentration of 0.5-1 mM. 12. Grow the protein overnight. Day 4 13. Collect a sample of induced bacteria. 14. Harvest bacteria by centrifugation, 5000rpm for 20 min at 4 degrees C. At this point you can freeze pellet at -80 degrees C for future purposes. 03/30/2009 Aleksandra Knapik |
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NU_native_expression | NU | NU_native_expression | expression | Proteins are expressed in E. coli in 1.5L of TB media supplied with Ampicillin and induced with 0.6mM IPTG for overnight at 25C. Cells are harvested by 10 min centrifugation at 8K rpm. Pellets resuspended in Lysis Buffer (43mM Na2HPO4, 3.25mM Citric acid, 250mM NaCL, 100mM Ammonium Sulfate, 5% Glycerol, 5mM Imidazole, 1.5mM Magnesium acetate, 1mM CaCL2, 0.08% DDM, 5mM b-mercaptoethanol) pH 7.8. Suspension then frozen at -30C. Cell extract for protein purification is obtained by sonication of previously frozen cell suspention in lysis buffer with follow up ultracentrifugation at 18K rpm, 4C for 40 min. |
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UVA_default_crystallization | UVA | UVA_default_crystallization | crystallization | Standard Virginia Crystalliation protcol: 1. After purification concentrate your sample to conc. 5-20 mg/ml using Amicon concentrators with proper MW membrane cut-off. It's better to prepare more concentrated samples because you can dilute it later 2. Aliquote your protein into samples of 10-50 ul and flash-freeze it in liquid nitrogen for future storage 3. Store your protein samples in -8- freezer in paper box 4. To set crystallization take fresh protein, or freezed one, then you need to thaw it on ice and then spin for 10 minutes, 3.000 rpm in 4C 5. Set screening crystallization using Mosquito Standard screens used for basic screening are: -Hampton Index -JCSG+ -SaltrX -PEGS -Cryo 6. Pipette selected screens from tubes into 96 well block, 1 ml in each well. Tape it well for future storage, put name of the screen, your name and date 7. Using multichannel pipetor pipette 200ul of each condition into mosquito plate 8. Take 5ul strips and put 5 ul of protein into each well 9. Set crystallization with Mosquito 10. Tape plates with foil 11. Enter plate into Crystal Trak, print barcode, choose apropariate observation schedule and put plate into Minstrel 12. Observe plate 13. Enter plate into Xtaldb 14. If you get crystalls, optimize conditions 15. Use hanging/sitting drop Linbrio or Nextal plates (you can't observe Nextal plates via Minstrel) 16. Enter plate into Xtaldb, create pipetting guide 17. Set crystallization 18. Observe plate 19. Harvest crystalls 07/20/2009 Aleksandra Knapik |
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UVA_default_crystal_harvesting | UVA | UVA_default_crystal_harvesting | crystal harvest | Standard Virginia Crystal Harvesting protocol: You will need: microscope, 2 dewars, small and big with liquid nitrogen, loops in vials, canes, wand, cryos, sample dish 1. Prepare loops - clean them and choose proper sizes 2. Sign vials and canes 3. Bring liquid nitrogen in 2 duars - small with vial's holder and big with long canes 4. Using microscope fish crystal from the well with loop 5. Immerse crystal in chosen cryo 6. Flash freeze crystal in liquid nitrogen 7. Put vial into proper position in cane (position 1 in cane is in the bottom) 8. Store frozen crystals in big dewar, remember to refill it with liquid nitrogen 07/20/2009 Aleksandra Knapik |
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Cloning_Toronto | UT | Cloning_Toronto | cloning | 1. PCR Amplification /Rxn dH2O 34.1 µl 10x Pfx buffer 5.0 µl 50 mM MgSO4 1.0 µl 10 mM dNTP’s 1.5 µl 10x Enhancer 7.5 µl genomic DNA (100ng/µl)0.4 µl Pfx (2.5U/µl) 0.5 µl Total Volume =50.0 µl (PFx enzyme and buffer from Invitrogen) *Make a master mix, aliquot 48 µl/rxn and add: 1.0 µl forward primer (50 µM) 1.0 µl reverse primer (50 µM) PCR Program: 1. 95°C 2:30 min. 2. 95°C 15 sec. 3. 53-56°C 30 sec. 4. 68°C 1 min./kb 5. Go To 2, Rep. 29x 6. 72°C 10 min 7. Hold 4°C 2. Run Large Gel – Verification of PCR product size 1% gel agarose gel in TAE Large : 3 g /300 ml Small: 0.5 g/ 50 ml 1.Heat in microwave 4-5 min (~1 min for small gel). 2.For large gels let the solution cool for 20 min before pouring the gel, for small gels cast right away and it will take ~20 min to solidify 3.Load 5 µl of PCR product with 1.5-2 µl of 6x SYBR green loading dye 4.Run gel @ 220-240 ~30 min. (large gel), @ 120V ~20 min. (small gel) 5.View gel using gel doc (Settings: Program as per lab settings) 3. PCR Clean-up For >12 PCR Products(QIAquick 96 PCR Purification Kit, handbook p. 20-22) 1.Transfer 45 µl PCR product to round bottom deep well (if the PCR reaction is 50 µl, there is not need to transfer into a new plate, PCR tubes/plate can be used). 2.Add 3:1 Buffer PM (135 µl). 3.Transfer to Qiagen Qiaquick 96 filter plate, pipette up/down a few times before transfer to mix sample and buffer. 4.Turn on vacuum for 5-7 min. 5.Add 900 µl Buffer PE, and vacuum for 5 min. 6.Repeat above. 7.Dispose of ethanol in waste tray, and vacuum 20-30 min. 8.Make sure all residual ethanol is gone by tapping on paper towel. 9.Elute with 50 µl EB buffer Or For <12 PCR Products(QIAquick PCR Purification, handbook p. 19-20) 1.Transfer 45 µl PCR product to 1.5 ml microcentrifuge tube. 2.Add 5:1 Buffer PB (225 µl), mix by pipetting. 3.Transfer to QIAquick Spin Columns (pink) and centrifuge @ 14,000 rpm for 1min. 4.Add 750 µl Buffer PE, centrifuge @ 14,000 rpm for 1min. 5.Discard ethanol and centrifuge again @ 14,000 rpm for 1min. 6.Transfer column to a clean 1.5ml microcentrifuge tube. 7.Let sit at room temperature for ~30 min. 8.Elute with 50 µl of EB buffer. 4. Ligation Independent Cloning (LIC)Infusion Reaction – Annealing vector and insert 1.Dissolve BD-Infusion pellet with 8.5 µl of prepared vector(p15Tv-L, see below). Use 2 µl of the infusion/vector mix and add to each insert, therefore each infusion pellet can be used for 4 reactions (for a 96-well plate, use 22 pellets that will be pooled and centrifuged before dispensing in PCR tube/plate. 2.Add 2-4 µl of PCR target to the vector and mix by pipetting up/down at least 3-4 times 3.Incubate for 30 min at 28ºC, then place reactions on ice or freeze until use. 5. Transformation 1.Place PCR tube/plate containing ligation on ice ~10 min. 2.Add 80 µl of competent DH5α to each ligation. 3.Leave on ice for 10 min. 4.Heat shock @ 42°C for 45 sec. 5.Place on ice for 2 min. 6.Recover in 900 µl of SOC for 1 hr. @ 37°C with shaking. 7.Spin down @ 3000 rpm for 10 min. 8.Discard supernatant (flip plate only, do not tap, ~110 µl of media should remain at bottom). Use 55 µl to plate on LB + ampicillin (100 µg/ml) + sucrose (5%) plates. (be sure to incubate plates at 37°C to allow proper absorption). 9. Incubate plates 22 – 24 h @ 37°C (to have colonies big enough to do the screen). 6. Colony Screen /Rxn H2O 16.2 µl Taq buffer (-MgCl2) 3.0 µl 25 mM MgCl2 2.4 µl 2 mM dNTP’s 3.0 µl 10x Enhancer 4.5 µl T7 promoter (~600 µg/ml)0.3 µl T7 terminator (~600 µg/ml)0.3 µl Taq (5 U/µl) 0.3 µl Total Volume= 30.0 µl Make a master mix, aliquot 28 µl/rxn and add colony from transfomration to each well of the PCR plate with a tip. Pipet up/down to mix the colony to the master mix. 2 colonies per target are screened PCR Program: 1.94°C 2 min. 2.94°C 30 sec. 3.50°C 30 sec. 4.68°C 1 min./kb 5.Go To 2, Rep. 29x 6.72°C 10 min 7.Hold 4°C 7. Run Large Gel – Verification of positive clones As previously reported (see aabove). 8. Grow 1.Pick positive colony for each clone, and add to 1 ml of LB + amp (100 µg/ml) in deep well plate. 2.Shake O/N @ 37°C. 9. Plasmid Prep of Positive Clones (QIAprep 96 Turbo Miniprep Kit, handbook p. 33-35) 1.Spin O/N grown plate for 10 min. @ 3000 rpm. 2.Discard supernatant. 3.Add 250 µl P1 buffer and vortex to resuspend pellet. 4.Add 250 µl P2 buffer (lysis buffer) and shake slowly, let sit for no more than 5 min. 5.Add 350 µl N3 buffer (neutralization buffer) and shake slowly. 6.Transfer (850 µl) to Qiagen Turbofilter plate (white), with Qiaprep plate (blue) beneath. 7.Vacuum for 3-5 min. 8.Discard TurboFilter, and place Qiaprep plate on top. 9.Vacuum for ~10 min. 10.Add 900 µl PE buffer, and vacuum 3 min. 11.Repeat above step. 12.Let sit at room temperature for ~30 min. 13.Elute into a shallow round bottom plate with 100 µl buffer EB. 14.Label plate and store at -20°C or aliquot plasmids Or For individual minipreps (Qiagen Manuel, pg. 22- 23) 1.Grow a colony of the positive clone in 2ml of LB+amp (100 µg/ml) in a 14 ml falcon tube overnight at 37°C. 2.The next day, centrifuge the tubes at 3000 rpm for 10 min. 3.Discard supernatant. 4.Add 250 µl P1 buffer and vortex to resuspend pellet. 5.Tranfer the sample to a 1.5ml microcentifuge tube 6.Add 250 µl P2 buffer (lysis buffer) and shake slowly,let sit for no more than 5 min. 7.Add 350 µl N3 buffer (neutralization buffer) and invert gently 8.Centrifuge for 10 min @ 13,000rpm 9.Apply the supernatants form step 8 to the QIAprep spin column by decanting or pipetting 10.Centrifuge for 1min @ 13,000rpm 11.Wash the column with 500 µl of Buffer PB and centrifuge for 1 min. 12.Wash the column with 750 µl Buffer PE and centrifuge for 1 min. 13.Discard the flow-through and centrifuge for an additional 1 min to remove the residual buffer. 14.Place the column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB and centrifuge for 1 min. 15.Label tube(s) and store at -20°C Preparation of p15TV-L vector please refer to this website for vector information http://www.thesgc.org/SGC-WebPages/StructureDescription/M&M/Vectors/p15TV-L.pdf 1.Transform p15TV-L in E. coli DH5α competent cells. 2.Grow 3 x 4 ml overnight cultures of p15Tv-L from single colony in LB + amp (100 µg/ml) in 14 ml Falcon tubes. 3.Miniprep using Qiagen spin columns (QIAprep Spin Miniprep Kit, handbook p. 22-23), using 4 ml of culture per column. 4.Elute with 50 µl of Buffer EB per column. 5. Digestion: p15Tv-L 120 µl 10x NEB #2or4 60 µl BseRI 15 µl H2O(RNAse/DNAse- free) 405 µl incubate 1.5 h @ 37C Add 10x NEB #2or 4 10 µl BseRI 5 µl H2O (RNAse/DNAse- free) 85 µl incubate 1.5 h @ 37C Total Volume =700 µl 6.Transfer to 15 ml conical falcon tube, add 3.5 ml of PB buffer 7.Purify by transferring 700 µl each to 6 Qiagen spin columns (QIAquick PCR Purification Kit, handbook p. 19-20). 8.Elute with 50 µl EB per column. Pool the samples and keep at -20oC until use. 9.Visualize digestion on 1% agarose gel (two bands should appear at ~8Kb and 2Kb). |
