Note

All centrifugation speeds are given in units of g.Please refer to Table 1 for information on vonverting g -force to rpm.If centrifuges/rotors for the required g -forces are not available,use the maximum g -force possible and increase the spin time proportionally.Spin until all liquid passes through the column.

All steps are carried out at room temperature.

Harvest Cells

Pellet 1-5 ml of an overnight recombinant E.coli culture by centrifugation.The optimal volume of culture to use depends upon the plasmid and culture density.For best yields,follow the instructions in the note below.Transfer the appropriate volume of the recombinant E.coli culture to a microcentrifuge tube and pellet cells at ≥12000 × g for 1 minute.Discard the supernatant.

Note: for best results with recombinant E.coli grown in LB (Luria Broth),use 1-3 ml of culture for high copy plasmids or 1-5 ml of culture for low copy plasmids.With recombinant E.coli grown in rich media such as TB (Terrific Broth)or 2 × YT,use only 1 ml of culture.Higher volumes can cause a reduction in yield.

1.Resuspend cells

Completely resuspend the bacterial pellet with 200 µl of the Resuspension Soulation.Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous.Incomplete resuspension will result in poor recovery.

Another rapid way to resuspend the cell pellets is to scrape the bottoms of the microcentrifuge tube back and forth 5 times across the surface of a polypropylene microcentrifuge tube storage rack with 5 × 16 holes.

2.Lyse cells

Lyse the resuspended cells by adding 200 µl of the Lysis Solution.Immediately mix the contents by gentle inversion (6-8 times)until the mixture becomes clear and viscous.Do not vortex.Harsh mixing wil shear genomic DNA ,resulting in chromosomal DNA contamination in the final revovered plasmid DNA .Do not allow the lysis reaction to exceed 5 minutes.Prolonged alkaline lysis may permanently denature supercoiled plasmid DNA that may render it unsuitable for most downstream applications.

3.Neutralize

Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution.Gently invert the tube 4-6 times.Pellet the cell debris by centrifuging at ≥12000 × g or maximum speed for 10 minutes.Cell debris,proteins,lipids,SDS,and chromosomal DNA should fall out of solution as a cloudy,viscous precipitate.If the supernatant contains a large amount of floating particulates after cengtrifugation,recentrifuge the suspernatant before proceeding to step 6.

4.Prepare Column

Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube,if not already assembled.Add 500 µl of the Column Preparation Solution to each miniprep column and centrifuge at 12000 × g for 30 seconds to 1 minute.Discard the flow-through liquid.

Note : The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.

5.Load cleared lysate

Transfer the cleared lysate from step 4 to the column prepared in step 5 and centrifuge at ≥12000 × g for 30 seconds to 1 minute.Discard the flow-through liquid.

6.Optional wash (use only with EndA+ strains)

Add 500 µl of the Optional Wash Solution to the column.Centrifuge at ≥12000 × g for 30 seconds to 1 minute.Discard the flow-through liquid.

Note: When working with bacterial strains containing the wild-type EndA+ gene,such as HB101,JM101,and the NM and PR series,the Optional Wash step is necessary to avoid nuclease contamination of the final plasmid DNA product.

7.Wash column

Important Reminder: Verify that ethanol has been added to the bottle of Wash Solution 2.

Add 750 µl of the diluted Wash Solution to the column.Centrifuge at ≥12000 × g for 30 seconds to 1 minute.The column wash step removes residual salt and other contaminants introduced during the column load.Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to remove excess ethanol.

8.Elute DNA

Transfer the column to a fresh collection tube.Add 100 µl of Elution Solution or molecular biology reagent water to the column.For DNA sequencing and other enzymatic applications,use water or 5 mM Tris-HCl,pH 8.0,as an eluant.Centrifuge at ≥12000 × g for 1 minute.The DNA is now present in the eluate and is ready for immediate use or storage at -20℃.

Note: If a more concentrated plasmid DNA preparation is required,the elution volume may be reduced to minimum of 50 µl.However,this may result in a reduction in the total plasmid DNA yield.

Results

Recovery and purity may be determined by spectrophotometric analysis.The ratio of absorbance at 260 nm to 280 nm (A260/280)should be 1.7-1.9.The size and quality of DNA may be determined by agarose gel electrophoresis or pulsed field electrophoresis.