Overview:
- In molecular biology laboratories, IPTG is commonly used to induce the expression of recombinant proteins in E. coli and to identify positive clones (blue/white selection) containing an insert in molecular cloning experiments. To be responsive to IPTG, the target gene must be under the control of lac or tac promoter/operator sequences.
- In absence of IPTG, lac repressor (lac I gene product) inhibits the transcription by binding to specific sequences, the lac operator, and inhibits the transcription of downstream genes.
- IPTG activates the transcription by binding and removing the lac repressor from the lac operator, thus allowing transcription of downstream genes by RNA polymerase.
- IPTG is commercially supplied as a white to off-white crystalline powder which is readily soluble in water. Generally, a 1 M IPTG solution in water is prepared that can be stored at -20°C for many months.
- 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.
Requirements:
Reagents
♦ IPTG powder
♦ Deionized / Milli-Q water
Equipment and disposables
♦ Falcon® Conical Tubes, 15 mL
Composition:
1M IPTG in water
Objective:
Preparation of 10 ml of 1 M IPTG solution
Preparation
Use appropriate personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow the guidelines of your institute.
Step 1: Weigh out 2.38 grams IPTG (Molecular Weight 238.3) and transfer to 15 ml Falcon® conical tube. Add 8 ml deionized / Milli-Q water. Tighten the cap and dissolve IPTG by inverting the tube several times.
Note:
IPTG dissolves quickly in water. Generally inverting the tube several times is sufficient to dissolve IPTG. Alternatively, one can use a magnetic stirrer to dissolve it.
Step 2: Adjust the volume 10 ml with deionized / Milli-Q water. Mix by inverting the tube a few times.
Step 3: Sterilize the solution by passing through a filter unit containing 0.22 µm filter paper.
Note:
Filter sterilization removes all suspended particles with a size of more than 0.22 μ which includes most bacteria and their spores but not mycoplasma. However, it does not inactivate enzymatic activities (e.g., DNases).
Precaution:
Never autoclave IPTG solution.
Storage:
The solution can be stored at -20 °C for several months in small aliquots (0.5 ml or 1.0 ml).
Applications:
♦ Protein expression and purification in E. coli
♦ Molecular cloning – Identification of positive clones using a blue-white selection
