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Protocol

Double Digestion |Gel Extraction|Miniprep | Chemical Inducible Expression of GFP |DNA Purification |Ligation of Insert DNA | PCR |Preparation of Competent Cells| Site-directed Mutagenesis| Transformation|

Miniprep Protocol & Hints

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Here is a suggested protocol; the yield of the plasmid should be approximately 0.2-0.3ug/ul. The bolded should be noticed for a nice miniprep.

Procedure:

1. Inoculate 5ml LB medium (containing antibiotic) with a bacteria l clone, culture with vigorous shaking at 37 degree for 12-16 hrs.

2. Put EB (elution buffer) or ddwater at 65 degree water bathing.

3. Harvest bacteria by spinning at 13000rpm (~12000g) for 1 min. Aspirate supernatant. Add additional 750 ul culture media, respin and aspirate supernatant for several times .

4. Resuspend bacterial pellet by complete vortexing in 250ml resuspension buffer(RB, with 10ul RnaseA in it), which should be stored at 4 ℃. The bacteria should be completely resuspended - noclumps should be visible.

5. Add 250ul freshly lysis buffer (LB) and mix gently by inverting 5-6 times at room temperature. The mixture should appear translucent and mucous-like. The time of lysis will never be longer than 5 min.

6. Add 350ul neutraliza tion buffer (NB) and mix gently by inverting 5-6 times, incubate at room temperature for 3 min. The mixture should contain flocculent white precipitate at this point.

7. Remove bacterial debris by centrifugation at 13000rpm for 10 min; pour supernatant to a fresh adsorption column which can avoid the transfer of precipitate to the new column causing the precipitate is "sticky".

8. Centrifuge at 13000rpm for 1 min. Pour off the liquid in the collection tube. For critical samples, repeat the operation above.

9. Add 650 ul washing buffer (WB) before centrifugation at 13000 rpm for 1 min.Pour off the liquid into beaker.

10. Centrifuge at 13000rpm for 10 min to spin the ethanol down.

11. Put the column into a fresh EP tube. Air dries DNA for 10 min.

12. Add 30-50 ul elution buffer (EB) to elute the DNA.

Tips:

1. Typica l yield of high-copy-number plasmids, such as PSB1AK3, prepared by this method is about 0.2-0.3 ug of DNA per ul of original bacteria l culture, and 0.1 ug of DNA per ul for low-copy-number plasmids such as PSB3T5.

2. To analyze the DNA by cleavage with restriction enzyme(s) remove 2 μl of the DNA solution and add it to fresh microfuge tube that contains 5 μ l of water. Add 1μl of the appropriate 10 x restriction enzyme (s). Incubate the reaction for 2 hr at the appropriate temperature. Store the remainder of the DNA preparation at -20 degree. Analyze the DNA fragments in the restriction digest by gel electrophoresis.

3. For tetracycline , notice its photolysis .

4. Resuspension buffer (RB) should be stored in the refrigerator. RNAse should be in the -20 degree freezer.

References:

• Current protocols in molecular biology