* Extremely simple to perform.
* You get up to 5 mg of DNA/-0.5L culture or ~95% of the amount present in the bacterial pellet.
* 260/280 ratio is ~1.8.
* It is very inexpensive in comparison to other vendors.
* Use Gloves while handling solutions for making plasmid preparations.

Resuspension Solution 200 ml Lysis Solution 200 ml Neutralization Solution 200 ml Suspension Solution 20 ml Precipitation Solution 40 ml Concentrated RNAse 250 µl 1X TE (Tris EDTA Buffer) 30 ml 10 X TE (Tris EDTA Buffer) 30 ml Gel Running Buffer (50 X TAE) 50 ml

Procedure:

1. Grow ~ 500 ml of bacterial culture at 37?C for 12-15 hrs with vigorous shaking. Pellet the cells by centrifugation at 5000 rpm for 10 minutes. Re-suspend the pellet in 10 ml of Re-suspension Solution. The pellet should be re-suspended thoroughly until no cell clumps are visible. Let the cell suspension stand at room temperature for 5 minutes.

Note: Growth of 500 ml culture of high copy plasmid may contain as high as 5 mg of plasmid DNA. Unlike column or resin based procedures, this procedure is designed to isolate entire amount of plasmid DNA from your culture. Therefore, depending upon your needs, you may scale up or down your preparations.

Suggestion: If a large amount of DNA prep is required but you do not wish to process at the same time, follow following simple steps:
Grow media in 1-liter culture. Re-suspend your pellet in 10 ml of TE buffer as a stock. Proceed with only 1/5th of stock pellet and save rest in a freezer (-20?C) for future use. Saving your stock of re-suspended pellet for future use will not affect the quality of DNA. Take 2 ml of re-suspended pellet and add 8 ml of Re-suspension solution. The rest of procedure is same.

2. Add 10 ml of Lysis Solution and mix the contents gently by inverting the tube 5-6 times. Let the solution stand for 5 minutes at RT. Do not use vortex to mix the contents.

3. Add 10 ml of Neutralization Solution and mix the contents by brief vortexing (5 seconds). Keep the solution in ice for about 10 minutes. Spin the tube at 12, 000 rpm for 20 minutes. The supernatant at this step should be clear with no visible clumps. Transfer the clear supernatant to a new tube by using a cheese cloth.

Note: If solution is not fully transparent, some precipitates may have gone into the filtered lysate. In that instance, spin the lysate for 10 minutes at 12,000 rpm and gently transfer the lysate to a new bottle.

4. Add 0.7 volume (21 ml) of Isopropanol to a bottle containing clear filtrate. Let the contents stand on ice for 5 minutes. Spin the tube for 10 minutes at 12, 000 rpm. The temperature during spinning could be set anywhere from 4 to 15?C. Discard the supernatant completely and spin the tube again (2-5 minutes) to remove any residual supernatant.

RNAse treatment

5. Dissolve the pellet completely in 1 ml of DNA Solublizing Solution. Add 10 µl of concentrated RNAse that is DNAse free. Let the tube stand for 10 minutes at room temperature. If the solution is not clear, spin the tube for 1 minute at maximum speed to remove any insoluble impurities. Transfer the clear solution equally into two microfuge tubes (2 ml capacity) as provided.

“Highly Super Coiled Plasmid DNA as made by Easy Pure Maxi Plasmid Preparation Kit"

DNA Purification

6. Add 1 ml of DNA Precipitation Solution in each tube. Mix the contents by brief vortexing and incubates tubes at room temperature for at least 10 minutes. Centrifuge the tube in a cold centrifuge for 10 min at maximum speed to precipitate the DNA.

Note: After incubation, it is safe keep the tubes in refrigerator (~4?C) until next day.

7. Remove the supernatant; rinse the pellet gently with 80% ethanol. Make sure that do not loose the pellet. Spin the tubes briefly for few seconds and carefully remove traces of ethanol wash without losing the DNA pellet. If you need plasmid DNA which is absolutely free of any salt, follow optional step (below), otherwise dissolve the DNA by using 0.75 ml of 1X TE. At this point the DNA pellet may be quite compact but place tubes containing DNA in a water bath (~42?C) for 10 minutes. Vortex or pipet the solution up and down to dissolve the DNA.

Optional step: Add 1 ml of 70 % ethanol and 100 µl of 3M sodium acetate (provided in 2 ml tube) in a tube containing DNA pellet. Vortex the tube tubes rigorously for 30 seconds. Keep tube in ice for 5 minutes. Pellet the DNA by spinning tubes in a cold centrifuge for 10 min at maximum speed. Carefully remove ethanol wash. Note: Prefer to transfer the ethanol wash into a new tube to avoid accidentally throwing away the DNA pellet.

Note: Take the spectrophotometric reading of DNA in a TE buffer. Avoid taking the reading in water as it can cause a shift in the OD due to an acidic pH of the water.