A stock solution (0.1 M) is prepared by dissolving IPTG in water with subsequent sterile filtration of the solution. The final concentration of IPTG in indicator plates should be 0.2 mM. Safety Information
- Dissolve 2.38 g of IPTG in 8 mL of distilled H2O.
- Bring to a final volume of 10 mL with molecular biology grade H2O.
- Filter sterilize with a 0.22 μ syringe filter.
- Store in 1mL aliquots at -20 °C.
What is the concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG) stock solution?
This protocol describes the preparation of a Isopropyl β-D-1-thiogalactopyranoside (IPTG) stock solution at various concentrations. A typical stock solution concentration is 100mM IPTG. A typical final concentration when using IPTG to induce protein expression under a lac operon is 0.1mM IPTG.
What is isopropyl β D thiogalactoside used for?
Isopropyl-β-D-thiogalactoside (IPTG) is commonly used in cloning procedures that require induction of β-galactosidase activity. It is used in conjunction with X-Gal or Bluo-Gal in blue-white selection of recombinant bacterial colonies that induce expression of the lac operon in Escherichia coli.
How do I prepare a stock solution of IPTG?
A stock solution (0.1 M) is prepared by dissolving IPTG in water with subsequent sterile filtration of the solution. The final concentration of IPTG in indicator plates should be 0.2 mM.
What are the ingredients in isopropyl β-D-1 thiogalactopyranoside stain?
Isopropyl β-D-1 thiogalactopyranoside (IPTG) Luria Broth (LB) powder or Tryptone, Yeast extract and NaCl Agar Sodium chloride (NaCl) Sodium phosphate dibasic (Na 2 HPO 4) Sodium Dodecyl Sulfate (SDS) Dithiothreitol (DTT) Glycerol Bromophenol Blue Glycine Tris base Coomassie protein gel stain Step 1. Reagents Antibiotic
How do you make 100mM IPTG solution?
IPTG – 1 M (100 x) Stock SolutionWeigh 2.383 g of IPTG. 2 g. ... Add 10 ml sterile H2O. Dissolve completely. ... Prewet a 0.2 µm syringe filter by drawing through 5-10 ml of sterile H2O and discard water. 00:02:00. ... Sterilize Ampicillin Stock through the prepared 0.2 µm syringe filter.
What is IPTG stock solution?
Introduction. This protocol describes the preparation of a Isopropyl β-D-1-thiogalactopyranoside (IPTG) stock solution at various concentrations. A typical stock solution concentration is 100mM IPTG. A typical final concentration when using IPTG to induce protein expression under a lac operon is 0.1mM IPTG.
How is IPTG prepared for induction?
Fast IPTG induction protocol Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37 C about 10min before use. After 3-4hrs remove 1ml from tubes at 37deg C and place in labeled 1.5ml tubes. Spin at max, 30sec, RT, and remove supe. Freeze pellet at -20 until needed.
How do you store IPTG stock solution?
0:092:08IPTG Stock Solution - YouTubeYouTubeStart of suggested clipEnd of suggested clipAnd the best way to store that stock solution to maximize its shelf life for the solid powder formMoreAnd the best way to store that stock solution to maximize its shelf life for the solid powder form of iptg. We recommend storing a desiccated at minus 20 this will give you the maximum product shelf-
How do I prepare for IPTG?
Stock Solution Preparation of 1 M IPTG (1 M IPTG Recipe)Dissolve 2.38 g of IPTG in 8 mL of distilled H2O.Bring to a final volume of 10 mL with molecular biology grade H2O.Filter sterilize with a 0.22 μ syringe filter.Store in 1mL aliquots at -20 °C.
What concentration of IPTG is used for induction?
While the optimal inducer concentration is 0.1 mM IPTG at 28 °C, it decreases at 34 and 37 °C to 0.05 mM IPTG. A transition area is visible at 30 °C, where 0.05 mM IPTG is preferable for early induction and 0.1 mM IPTG is better for later induction.
How is IPTG calculated?
When your required O.D. value for the culture is reached, add 1mL of this stock to 1 litre of broth culture so that the final concentration of IPTG is 1mM. This is according to the formula C1V1= C2V2 (C: concentration, V: Volume) which you must be already using in general for all solution preparations.
What happens if you add too much IPTG?
yes IPTG halts the divison process but enhances protein production. however, if we increase IPTG beyond a limit the divison of bacteria is compromised and which in turn effect the protein machinery of the cells. Yes, a high concentration of IPTG is toxic to the cell.
How long does IPTG last at 4c?
IPTG should be stored immediately upon receipt at –20°C. IPTG is stable for at least 9 months when stored unopened at –20°C.
Does IPTG need to be kept on ice?
IPTG solutions can be stored at room temperature for up to one month. Maximum IPTG product life can be achieved by avoiding repeated thaw/freezing.
Where are IPTG stored?
IPTG powder can be stored at Room Temperature. For optimal shelf-life, we recommend storage, with dessicant, at -20oC. IPTG stock solution is typically made at a 1M concentration and stored at -20oC. We recommend making single-use aliquots to prevent repeated freeze-thaw cycles of IPTG in solution.
What is the formula weight of IPTG?
238.31 g/molIsopropyl β-D-1-thiogalactopyranoside / Molar mass
Application
IPTG is commonly used in cloning procedures that require induction of β-galactosidase activity. It is used in conjunction with X-Gal or Bluo-Gal in blue-white selection of recombinant bacterial colonies that induce expression of the lac operon in Escherichia coli.
Reconstitution
A stock solution (0.1 M) is prepared by dissolving IPTG in water with subsequent sterile filtration of the solution. The final concentration of IPTG in indicator plates should be 0.2 mM.
General description
Isopropyl-β-D-thiogalactoside (IPTG) is a chemical analog of galactose that cannot be cleaved by β-galactosidase. IPTG functions by binding to the lacI repressor and altering its conformation, which prevents the repression of the β-galactosidase coding gene lacZ.
Application
Isopropyl-β-D-thiogalactoside (IPTG) is commonly used in cloning procedures that require induction of β-galactosidase activity. It is used in conjunction with X-Gal or Bluo-Gal in blue-white selection of recombinant bacterial colonies that induce expression of the lac operon in Escherichia coli.
Specifications
Specific Activity: The described standard assay will results in a specific activity of 1.8 x 109 dpm/μg, corresponding to 65% incorporation with different substrate DNAs in 30minutes. When varying the ratio of template DNA to labeled dNTP, similar incorporation rates, but different levels of specific activity of the labeled probe are obtained.
Preparation Note
Working concentration: Final concentration should be 0.2 mM. Storage conditions (working solution): Stock solutions of the product in water (e.g., 1 M IPTG) stored at -15 to -25 °C, are stable up to six months.
Application
IPTG is commonly used in cloning procedures that require induction of β-galactosidase activity. It is used in conjunction with X-Gal or Bluo-Gal in blue-white selection of recombinant bacterial colonies that induce expression of the lac operon in Escherichia coli.
Reconstitution
A stock solution (0.1 M) is prepared by dissolving IPTG in water with subsequent sterile filtration of the solution. The final concentration of IPTG in indicator plates should be 0.2 mM.
Articles
General protocols for growth of competent cells and their transformation (uptake of DNA).
Frequently Asked Questions
What Is The Protocol For The Extraction of Protein from Bacteria?
- Fast Induction (does not work for all, can give suboptimal yields):
1. From a relatively fresh plate (<4 weeks), pick a colony and grow O/N at 30°C (or 37°C) in 1-2 mL LB+AMP (or other selection) in a 15 mL snap cap tube on a rotator or shaker. 2. Dilute 1:50 (1:100 if 37°C O/N) in 2 mL LB+AMP and grow 3-4 hours at 37°C in 15 mL snap cap tube in a rotator. 3. … - Slow Induction (can enhance solubility of proteins):
1. Add 20°C 1 mL LB+AMP+1mM IPTG to 15 mL snap cap tube and incubate rotating or shaking at 20°C for 12-16 hours. This will get the final volume back to 2 mL and the final concentration of IPTG to 0.5 mM. 2. After 12-16 hours, transfer 1 mL from induced sample to labeled 1.5 mL tube…
Additional Reading
- IPTG Triggers the Transcription of the lacOperon
- IPTG in New Research: sRNAs & Alternative Inducers
- IPTG in Synthetic Biology: Top 3 Review Papers