Incubate induced cells for 4 to 24 hours and harvest using preferred method. Beta-Galactosidase Activity Assay 3. Equipment and disposables Isopropyl-β-D-Thiogalactopyranoside (IPTG) Dissolve 2 g of IPTG (m.w. Add 10 µl IPTG (100mM) per 1 mL of Media for a final concentration of 1mM. Sterilize by filtration, then store at -4¡ãC. But usually we use 0.5mM IPTG concentration, only when OD (600) of bacterial cells reached up to 0.6-0.8. 0,5ml of a 1M stock solution) and appropriate antibiotics) to 1 liter of autoclaved media agar, just prior to pouring the plates. 1. How long does IPTG last? - Gaming Section : Magazine ... Incubate overnight at 30°C. PDF Promoter Selection Kit 100 µl IPTG stock per 1 L agar solution, dilute 1:1000 to soak nitrocellulose membranes for library screening) Return to: Home - LabLinks - GH-PRL-PL. Contains no animal-derived products. Filter-sterilize and store at room temperature. Typically used in conjunction with IPTG for blue/white screening via the lac operon. PDF IPTG Induction and Extraction of Proteins Protocol v3 Add to bookmarks. Autoclavable. After 3-4 hrs remove 1 mL from tubes at 37°C and place in labeled 1.5 mL tubes. 4. 58 L of 1M IPTG is added to the conical flask containing the remaining 58mL of bacterial culture. Chemistry. How much water and how much IPTG (in grams) will need to make a sufficient 1M stock solution? 2. Add 100µL of high titer bacteriophage stock (>108 pfu/mL) (the volume can be adapted according the titer). - Get the answer to this question and access a vast question bank that is tailored for students. 4) Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37 C about 10min before use. o Ensure that the bacterial cells are still growing after addition of IPTG (by monitoring OD readings) → important to ensure that the addition of . psb1A3 VS45 cells were grown on Chloramphenicol and Amp combined plated, with 20% w/v Arabinose and 1mM IPTG plate. IPTG concentration (0.1mM - 1.0mM) is very effective for protein expression. 16. Note: To prepare 10 mL 1M IPTG stock solution, weight 2.38 g IPTG and dissolve in 8 ml distilled water Used in the stimulation of β-galactosidase in cellular systems in which dioxane would disrupt normal cell function. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+LacH containing bacterial cultures (with appropriate antibiotic) 2. How to make a IPTG - 1 M (100 x) Stock Solution . Is IPTG soluble in water? Grow at 37°C all day. 1M MgSO 4 (sterile filtered) 2M CaCl 2 (sterile filtered) 0.5% (w/v) Thiamine Solution (sterile filtered) Procedure Day 1. Spin at max, 30sec, RT, and remove supe. How do you prepare a 1 M HCl solution, which has a specific gravity of 1.18? Filter sterilize and store at 4°C. Follow Qiagen miniprep protocol up to the addition . Prepare two stock solutions for the next day: PBS 1X, pH=7 (Solution 1) and PBS 1X (pH=7), glucose (5 g/L), phenylpyruvate (0,5 g/L) (Solution 2). . Calculate the volume required to prepare the solution. Make 100 ml by combining 2 ml of 1 M HEPES, pH 7.9 stock solution, 1 ml of 2 M KCl the synthetic promoters tac and and trc, and the arabinose inducible , and the arabinose inducible ara. or buy 25 mL 1M stock solution Teknova 13431 (Fisher) Store at -20C 2 Buffers and stock solutions for western blot Recipes for western blot buffers and stock solutions - RIPA buffer (radioimmunoprecipitation assay buffer) - Nonidet -P40 (NP 40) buffer - Cytoskeletal bound protein extract buffer - Soluble protein buffer - Sodium orthovanadate preparation - TBS 10X (concentrated Tris-buffered saline) - TBS 10X alternative recipe (concentrated Tris . Filter sterilize (0.2µm), and store in 5ml aliquots at -20°C. 2. Isopropyl-β-D-Thiogalactopyranoside (IPTG) Dissolve 2 g of IPTG (m.w. 1. Answer (1 of 6): By definition … \text{Molarity of solution}=\dfrac{\text{moles of solute}}{\text{volume of solution in litres}} …the product… \text{Molarity of solution}×\text{volume of solution in litres}=\text{moles of solute} And thus for a 1•L volume of NaOH(aq) at 0.10•mol•L^{-1} concen. 1) 6L of 1mM IPTG. Day 1. ), and a single E.coli colony. Filter sterilize (0.2µm), and store in 5ml aliquots at -20°C. LAB 3: CLONING Methodology Prelab Preparation Preparation of X-GAL and IPTG 1) IPTG Dissolve 1.2g IPTG (isopropyl-β-D-thiogalactopyranoside) in deionized water to a final volume of 50ml. 14. Note: To prepare 10 mL 1M IPTG stock solution, weight 2.38 g IPTG and dissolve in 8 ml distilled water, adjust to a final volume of 10 ml with distilled water. ZnCl 0.1M 250 ml: 3.4 g ZnCl. Spin down the remaining volume of cells in 15ml conical tube. LB Medium 10g Bacto®-tryptone MgCl2 1M 500 ml: Add 101.65 g MgCl2.6H2O into 500 ml ddH2O. Adjust the volume of the solution to 10 ml with H 2 O and sterilize by passing it through a 0.22-µm disposable filter. From a relatively fresh plate (<4 weeks) pick a colony and grow O/N at 30C (or 37C) in 1-2ml LB+AMP (or other selection) in a 15ml snap cap tube on a rotator or shaker. Ready-to-apply ChromoMax IPTG/X-Gal solution is a proprietary formulation that allows you to skip the cumbersome preparation process that traditionally requires tedious weighing and dissolving of fine powder in toxic solvents such as DMSO or DMF. Is IPTG soluble in water? Induce expression with 1mM [IPTG]final [IPTG]final = 1 mL 1M IPTG/L culture ; Aliquot into 1mL in 1.7 mL Eppendorf tubes ; Grow at 37°C for 3-4 hours Finish around 4PM ; Take sample (1 mL) of cells, pellet at 21,000 xg for 1 minute ; Resuspend pellet (1 mL sample) in 50 μL of denaturing sample buffer. Freeze pellet at -20°C until needed. Filter-sterilize (0.2 µm) and store at 4º C IPTG stock solution, 0.1M 1.2g IPTG (Cat.# V3955) Add water to 50ml final volume. The final concentration of IPTG is 0.1M. (dilute 1:10000 for indicator agar plates, e.g. Agar Ampicillin. 3. Dilute 1:50 (1:100 if 37C O/N) in 2ml LB+AMP and grow 3-4 hours at 37 C in 15ml snap cap tube in a rotator. SOLID NaOH BURNS YOUR SKIN BUT YOU MAY NOT FEEL IT AT FIRST! Adjust the volume of the solution to 10 ml with distilled H 2O and sterilize . Filter sterilize with 0.22 um membrane cartridge. How much water and how much IPTG (in grams) will you need to make a sufficient 1M stock solution? Cell Resuspension Solution 25mM Tris-HCl (pH 8.0) 10mM EDTA 50mM glucose IPTG Stock Solution (0.1M) 1.2g isopropyl β-D-thiogalacto-pyranoside (IPTG) (Cat.# V3951) Add deionized water to 50ml final volume. Thermo Scientific IPTG (isopropyl-beta-D-thiogalactopyranoside) is a highly stable synthetic analog of lactose. Description. A typical final concentration when using IPTG to induce protein expression under a lac operon is 0.1mM IPTG. Dissolve: the molecular weight is 238.3, so this is 0.238 g in 10 ml of water. Preparation of X-Gal/IPTG LB Agar Plates for Blue/White Colony Screening For individual LB (Luria Broth) agar plates: 1. 50x Glucose (150-ml Stock Solution) D-Glucose 2 M 54 g Dissolve and adjust to 150 ml with ultrapure H 2O. Prepare amino acid 50x Stock. Dry opened LB plates at room temperature under UV light for about 30 minutes. How to make a IPTG - 1 M (100 x) Stock Solution . Dilute and autoclave before use. Sterilize through a 0.2 µm membrane filter and dispense in 2-3 mL aliquots in sterile vials or tubes. A 5X stock solution is prepared by dissolving 54 g Tris base, 27.5 g Boric acid, and 20 ml of 0.5 M EDTA in water to a final volume of 1000 ml. Make the final volume to 30ml and mix again. 0.2N NaOH, 1% SDS in deionized water. 2) Dilute 1:50 (1:100 if 37C O/N) in 2ml LB+AMP and grow 3-4 hours at 37 C in 15ml snap cap tube in a rotator. 1. It inactivates the lac repressor and induces synthesis of beta-galactosidase, an enzyme that promotes lactose utilization. The 5X concentrated solution contains 0.445 M Tris borate and 0.01 M EDTA (pH 8.2 - 8.4). Directions: 1) Mix 238 mg of IPTG with 900 µl of ddH 2 O.. 2) Add ddH 2 O until final volume is 1 ml.. 3) Store at -20°C. Dispense the solution into 1-ml aliquots and store . Prepare M9 stock media. Safety Information Storage Class Code 11 - Combustible Solids WGK WGK 3 Flash Point (F) Not applicable Flash Point (C) Not applicable Personal Protective Equipment The stock solution of IPTG is stable at this temperature for 2-4 months. Add a final concentration of 2mM of CaCl2 and MgCl2 (for example add 70µL of CaCl2 1M and 70µL of MgCl2 1M in the 35mL culture). The molecular weight of IPTG (CAS 367-93-1) is 238.3 g/mol which means you need to dissolve 238.3 grams of IPTG in water to prepare 1 L of 1M solution. Responsible: Matheus Araujo Description: The IPTG stock was done from 1,19g of lyophilized IPTG and 5ml of nuclease-free water, filtered and stored at -80 graus C. A 1M Sucrose solution also was done, followed by the TSE solution. value for the culture is. Prepare all the buffers described in Step 1, except make fresh IPTG stocks. LB medium with ampicillin To 1L of distilled water, add: 10g Bacto®-Tryptone 5g Bacto®-Yeast Extract 5g NaCl Adjust the pH to 7.5 with NaOH. After IPTG induction, the E.coli BL21 transferred with pGEX-4T-2 vector produced 26kDa GST protein (lane2) and E.coli BL21 transferred with pGEX-4T-2-BTP-1M produced 44kDa protein matched well with our GST-BTP-1M fusion protein (lane 4). Allow the auto-claved medium to . Recover overnight cultures. Inducer of β-D-galactosidase. 0.1 M NaOH solution . Start of the day: To 3 cultures from Day 1, Step 1 add to each 15 ul of 1M IPTG . Spin at max, 30sec, RT, and remove supe. sterile filter and store at -20°C. combining 10 ml of 1M HEPES, pH 7.9 stock solution, 5 ml of 2 M KCl stock solution, 1 ml of 0.5 M EDTA stock solution, and 484 ml of H 2 O. Add the following to 800ml H2O 10 g Bacto-tryptone- (1% w/v) 5 g yeast extract-(0.5% w/v) 10 g NaCl-(1% w/v) 2. Filter sterilize with syringe and 0.22μm filter Alternatively Dissolve 238 mg IPTG (sigma I-6758) in 10 ml DW for (100mM stock) store in 1 ml aliquotes at -20°C Generally a 1mM solution is an effective amount to induce the pLac promoter region. Add single colony or glycerol stock to 5ml LB with 25ug/ml chloramphenicol in addition to any resistance markers on plasmids Shake at 37C for 12-14 hours overnight Autoclave 1L LB flasks Add entire contents of overnight cultures to 1L flasks (in addition to antibiotics) and shake at 37C Grow to an OD of ~0.5 and induce with 1ml of 1M IPTG 3 . Isolation of bacmid DNA . Run. 1m 12h 5m 4m 10m 5 IPTG stock solution (1 M) 0.25 g of IPTG 1049 uL of DI water filter sterilize with .22 um filter. 1.0M IPTG stock** § Vendor: Anatrace, Cat# I1003, FW=238.31 § Dissolve 7.15gm of IPTG in 20ml of ddH 2 O and vortex it for some time. After 3-4hrs remove 1ml from tubes at 37deg C and place in labeled 1.5ml tubes. Freeze pellet at -20 until needed. A typical stock solution concentration is 100mM IPTG. Prepare a glycerol stock of each recombinant bacterial clone by mixing 0.5 ml of overnight culture with 0.2 ml of sterile 50% glycerol in a cryovial. IPTG is widely used to induce the expression of cloned genes under the control of the lac operon and to screen blue/white colony in X-gal plate. 2. It is used in conjunction with X-Gal (#R0941) to determine the lac phenotype . Preparation of Reagents. Prepare a 2mL day culture consisting of 2mL LB media, 2uL antibiotics (1000x conc. 100 mg/mL IPTG. The T7 IPTG-inducible system has become the dominant expression system currently employed. add 2.5 ml of 10 mg/ml ampicillin stock and 100 µl of 1M. Copy / Fork. For longer periods, store at -20ºC. However, the use of a strong promoter, which leads to hyper-expression levels, is not without potential adverse consequences. Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37 C about 10min before use. Requirements: Reagents ♦ IPTG powder ♦ Deionized / Milli-Q water. Centrifuge for 20 minutes at 4500 rpm and 4°C. THIS IS THE UNINDUCED CONTROL. The chemical formula is here. 13. Autoclavable. ), and a single E.coli colony. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+pBAD containing bacterial cultures (with appropriate antibiotic) Day 2. Once the cells grew 25µl of 1x PBS were pipetted onto a spot of bacterial cells, the PBS was . Dissolve 1g in 4196 μL deionized water to make 1M solution. pStock Solution Concentration: 0.1 Mp. 1M IPTG stock solution supplemented M9 minimal media 15ml falcon tubes Preparing IPTG stock solutions. Filter-sterilize (0.2µm) and store at 4˚C. Store 10X stock at 4 °C from which you can dilute 1:10 to make 1X working stock to keep at room temp. THIS IS THE UNINDUCED CONTROL. Adjust the volume of the solution to 5 ml with H2O and sterilize by passing it through a 0.22-µm disposable filter. Print. Store glycerol stocks at -80°C. IPTG is widely used to induce the expression of cloned genes under the control of the lac operon and to screen blue/white colony in X-gal plate. Ready-to-apply ChromoMax IPTG/X-Gal solution is a proprietary formulation that allows you to skip the cumbersome preparation process that traditionally requires tedious weighing and dissolving of fine powder in toxic solvents such as DMSO or DMF. 1. Note: To prepare 10 mL 1M IPTG stock solution, weight 2.38 g IPTG and dissolve in 8 ml distilled water, adjust to a final volume of 10 ml with distilled water. MAKE SURE ANY SPILLS ARE CLEANED UP VERY WELL! 4) Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37 C about 10min before use. AFM Preparation procedure. Dilute and autoclave before use. Grow at 37°C all day. Export. View this method on protocols.io and use it directly at your bench Comparable Items: 15. A stock solution (0.1 M) is prepared by dissolving IPTG in water with subsequent sterile filtration of the solution. Adjust the volume of the solution to 10 ml with distilled H 2O and sterilize . Add 12,5 uL of IPTG (1M) to the cultures. IPTG: isopropyl thiogalactoside, or isopropyl beta-D-thiogalactopyranoside. 5. Inoculate with 25 to 50µl of the glycerol stock. The HCl solution has 35% purity. Prepare a stock solution in the range of 0.1M - 1M (1000x) using ultrapure water and filter sterilize. Prepare a 2mL day culture consisting of 2mL LB media, 2uL antibiotics (1000x conc. Store at -20 ˚C. Note: IPTG concentration can vary from 0.1 to 1M. When your required O.D. Adjust pH to 7.5 with NaOH. 1) Take out an IPTG 1M aliquot from -20°C Freezer and wait to thaw 2) Create 2 diluted solutions: 0.1M IPTG- add 100ul of 1M IPTG to 900ul of sterile H2O 0.01M IPTG- add 10ul of 1M IPTG to 1ml of sterile H2O. Incubate at 37°C with agitation (200rpm) a few hours or overnight.*. Freeze pellet at -20 until needed. Begin of expression cultivation. Dissolve 1.0 g of isopropylthio-ß-D-galactoside (IPTG) in 8.0 mL of deionized water. 4. Store in 1mL aliquots at -20 °C. You need to prepare a stock solution of IM IPTG that will be sufficient to give you 6L of ImM IPTG once it is diluted. Generally, a 1 M IPTG solution in water is prepared that can be stored at -20°C for many months. Prepare M9 stock media. Mix. Spread 100µl of 100mM IPTG on the above plate (LB/Amp) and then spread 20ul of 50mg/ml X-Gal. Filter-sterilize and store at room temperature. 2. 5) After 3-4hrs remove 1ml from tubes at 37deg C and place in labeled 1.5ml tubes. Sterile the solution using a 0.22 um syringe filter. Record the optical density at 420 nm and at 550 nm for each tube. THIS IS THE UNINDUCED CONTROL. Dilute 10x to make 100 mM CaCl2. Spin at max, 30 sec, room temperature, and remove super. Chemistry questions and answers. 5; Calculate the units of activity 6. You need to. The solution is sterilized by autoclaving and is stored at room temperature. Fast IPTG induction protocol. - Note time of addition precisely. C 2 = 0.001M; V 2 = 58mL → 700 L + 70mL - 1mL - 1mL ≈ 58mL ∴ V 1 is calculated to be 58 L i.e. You need to prepare a stock solution of 1M IPTG that will be sufficient to give you 6L of lmM IPTG once it is diluted. Add 40 μL of the Thermo Scientific X-Gal Solution (20 mg/mL), ready-to-use (Cat # . protocols.io. Centrifuge at maximum speed for 30 seconds at room . Prepare amino acid 50x Stock. Adjust the volume to 10.0 mL with deionized water. Thermo Scientific IPTG (isopropyl-beta-D-thiogalactopyranoside) is a highly stable synthetic analog of lactose. Preparation of M9: Weigh appropriate amount of Na2HPO4, KH2PO4, NaCl, NH4Cl, glucose and add to 950 ml of H2O one by one while stirring solution with a magnetic bar. Bring to a final volume of 10 mL with molecular biology grade H 2 O. Filter sterilize with a 0.22 μ syringe filter. New version. Generally, M9 salts contain a nitrogen source in the form of NH 4 Cl. ABE: 20 mM HEPES, pH 7.9, 20 mM KCl, 1 mM EDTA, 10 mM maltose. - Prepare a 150mL overnight culture consisting of 150mL LB, 150uL antibiotics (1000x conc. IPTG Dissolve 1.2g IPTG (isopropyl-β-d-thiogalactopyranoside) in deionized water to a final volume of 50ml. 3. A typical stock concentration of X-gal is 50mM (20mg/mL).To apply X-gal directly to the top of agar plates for blue/white screening, apply 40μL and wait to dry. Make PBS-T by supplementing with 0.05 - 0.1% Tween20. Description. Return cultures to either 25 °C or 30 °C shaking incubator 5. Aliquot and store at room temperature or 4ºC for up to 1 month. It inactivates the lac repressor and induces synthesis of beta-galactosidase, an enzyme that promotes lactose utilization. Since we want to add a labeled nitrogen source . X-gal: 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Agar Media (Recommended) 1. IPTG is used to induce expression of cloned genes under control of the lac operon. Preparation of LB-Ampicillin IPTG Agar This protocol is used to prepare solid LB-ampicillin IPTG (Isopropyl b-D-1-Thiogalactopyranoside) media which will select for the growth of bacteria transformed with plasmids containing an . How to make a IPTG - 1 M (100 x) Stock Solution . Prepare 1 ml LB + Antibiotic + 1mM IPTG in a 15 ml conical and prewarm to 37°C about 10 minutes before use. MgSO4 1M 500 ml: Add 123.3 g MgSO4.7H2O into 500 ml ddH2O. Stock Solution Preparation of 1 M IPTG (1 M IPTG Recipe) Dissolve 2.38 g of IPTG in 8 mL of distilled H 2 O. 20 g of glucose 50 mL of DI water filter sterilize. This plate contained both PVS72 and PVS105 cells as controls, it was then incubated for 5 days at 22 o C . Stop the reaction after sufficient yellow color has developed 3 by adding 0.5 mL 1M Na 2 CO 3 4. Make stock solution of IPTG by dissolving 1 g of IPTG in 4 ml of distilled H 2 O. control for gel analysis and glycerol stock (see Glycerol stocks), once this culture has grown for 6 to 8 hours. Prepare a 1M stock of IPTG (using the Molarity formula) and store at -20 degress centrigrade and away from light ( IPTG is light-sensitive). IPTG, Animal-Free, High Purity - CAS 367-93-1 - Calbiochem. Filter-sterilize through a 0.2µm filter unit and store at 4°C. = 238.3 g/mole) in 8 ml of distilled H 2O. IPTG (stock solution, 200mg/ml) IPTG is isopropylthio-D-galactoside. Add 1.0 mL per liter ofsterilizedmedium that has cooled to 55 ˚C. 0.1M EDTA stock § EDTA disodium salt § Vendor: Fisher, Cat# BP120-500, FW=372.24 § Dissolve 37.22gm of EDTA in ddH 2 O and make the volume to 1L. Prepare fresh for each use. Stock Solution Concentration: 20 mg/ml 0.1 mM IPTG (some recipes say 0.5 mM) 40 µg/mL X-gal (alternatively 50 µl of 50mg/ ml X-gal and 100 µl of 0.1M IPTG can be spread on previously prepared LB plates) IPTG stock solution (0.1M) 1.2g isopropyl-B-D-thiogalactopyranoside (IPTG) Add deionized water to 50 ml final volume. 5) After 3-4hrs remove 1ml from tubes at 37deg C and place in labeled 1.5ml tubes. IPTG is used to induce expression of cloned genes under control of the lac operon. 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And store at 4°C ( 0.1M ) 1.2g IPTG add Milli-Q water to final... Either 25 °C or 30 °C shaking incubator 5 CAS 367-93-1 - Calbiochem incubated for days. Lb agar ( about 25 ml ) into a Petri dish, not. Lb, 150uL antibiotics ( 1000x conc in step 3 and place in labeled 1.5ml tubes remaining volume of mg/mL. Iptg/X-Gal solution 1ml ; 10... < /a > AFM Preparation procedure in cellular systems in which dioxane would normal! Fw 147 ), and remove supe is a highly stable synthetic analog of lactose transformant... 3-4 hours, remove 1 ml from tubes at 37°C and 1m iptg stock preparation in labeled 1.5ml tubes ( with antibiotic... Remaining volume of the lac phenotype sterilize through a 0.2 µm membrane filter and dispense in 2-3 aliquots. Of 1mM IPTG step 1 add to each 15 ul of 1M IPTG reached up to 1.... A lac operon is 0.1mM IPTG IPTG last 1M IPTG to induce expression of cloned genes under control of lac. Reached up to 0.6-0.8 stock solution harvest using preferred method concentration when IPTG. Test culture for induction for a final volume of the solution using a 0.22 μ syringe.. ( m.w to this question and access a vast question bank that is tailored for students to 25! A 0.22-µm disposable filter - Quora < /a > Preparation of Reagents solution is sterilized by autoclaving and stored. Conical and prewarm to 37 C about 10min before use ofsterilizedmedium that cooled! Culture consisting of 150mL LB, 150uL antibiotics ( 1000x conc containing bacterial cultures ( with antibiotic... Before using it to spread your transformant bacteria vary from 0.1 to 1M of water will to... Add 58.8 g CaCl2.2H2O ( FW 147 ), ready-to-use ( Cat # used in conjunction with for. With 20 % w/v Arabinose and 1mM IPTG plate chemicals for 30min/37C before using to... < /a > 1 how much IPTG ( m.w 5 ml with H 2 filter... G in 10 ml with deionized water to 50ml final volume to 10.0 ml with H2O and sterilize ml! Volume per litter... < /a > Description but you MAY not FEEL it at FIRST,., only when 1m iptg stock preparation ( 600 ) of bacterial cells, the use a... Tube, 1m iptg stock preparation 5 & # x27 ; at maximum speed for 30 seconds at room temperature or 4ºC up... Is stored at -20°C //www.researchgate.net/post/How_much_concentration_of_IPTG_and_its_volume_per_litter_will_be_use_for_protein_induction '' > Fisher BioReagents ChromoMax IPTG/X-Gal solution 1ml ; 10... /a. With 25 to 1m iptg stock preparation of the day: Prepare and incubate ( )! In indicator plates should be 0.2 mM for 3-4 hours, remove 1 from... Iptg concentration, only when OD ( 600 ) of bacterial cells, the PBS was Amsterdam/extra/protocols! Dissolving 1 g of IPTG by dissolving 1 g of isopropylthio-ß-D-galactoside ( IPTG ) dissolve g. Large 2 L Erlenmeyer flask ) water filter sterilize ( 0.2µm ), ready-to-use ( #... Need to make 1X working stock to keep at room temperature under UV light for about 30 minutes LSM2191! Cloned genes under control of the day: Prepare and incubate ( 37C ) 6x 3ml pSB1AT3+pBAD containing bacterial (...