How to prepare 1M IPTG solution for protein expression induction?
引言
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
- 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.
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Step 2: Adjust the volume 10 ml with deionized / Milli-Q water. Mix by inverting the tube a few times.
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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