Sample Induction Protocol(for reference only)

1.   Inoculate a single colony from a freshly streaked plate into 3ml of LB medium containing the appropriate antibiotic for the plasmid ａnd host strain.

2.   Incubate with shaking at 200 rpm at 37℃ overnight. 

3.   Inoculate 50 ml of LB medium containing the appropriate antibiotic with 0.5 ml of the overnight culture prepared in step 2(use the 500 ml triangular flask as the container would be better).

4.   Incubate with shaking at 150 rpm at 37℃ until the OD 600 reaches 0.5-0.8. (0.6 recommended; about 2.5h).

5.   (Optional)Pipet 1ml of  the cultures into clean microcentrifuge tubes and  place the tubes on ice until needed  for gel analysis or storage at  -20℃. These will serve as the non-induced control samples.     

6.   Add IPTG to a final concentration of 1 mM.  Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.

7.   Incubate with shaking at 120 rpm at 37℃ for 2-4 hours. To determine the optimal time for induction of the target protein, it is recommended that a time course experiment be performed varying the induction from 2-16 hours.

8.   Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10

minutes at 4℃.

9.   Remove the supernatant ａnd store the cell pellet at -20℃ (storage at lower temperatures is also acceptable). 

 

IPTG

Prepare a 1 M solution of IPTG (Isopropyl-β-D-thiogalactoside; Isopropyl-β-D-thiogalactopyranoside) by

dissolving 2.38 g of IPTG in dd water ａnd adjust the final volume to 10 ml. Filter sterilize before use. 

氯霉素

Chloramphenicol  34 mg/ml in ethanol. Store at –20°C.  Use at 34 µg/ml.

 

 

注意事项

1. 感受态细胞在冰上融化。

2. 混入质粒时应轻柔操作。

3. 转化高浓度的质粒可相应减少*终用于涂板的菌量。

4. 诱导时，IPTG浓度可选（0.1-2mM均可）。

5. 为获得需要量的蛋白，*佳诱导时间，温度，IPTG浓度需实验者优化。

6. BL21(DE3)pLysS 菌株携带 pLysS质粒，除复苏培养基为无抗生素外，其余所用培养基、培养液均应含有34 µg/ml氯霉素，以防质粒丢失。

7. 具有卡那霉素抗性，不能用于具有卡那霉素抗性质粒的表达。