Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

The overall goal of this procedure is to use a streamlined method to purify plasmid, DNA from e coli. This is accomplished by first growing overnight cultures in a deep well plate. Then harvest and lice the bacterial cells.

Next, following precipitation of protein and genomic DNA, clarify, the cell lysates finally isolate plasmid DNA on the DNA binding plate. Ultimately, this system offers a higher yield of sequence quality DNA using a cost effective procedure. The primary advantage of this technique over pre-packaged kits is this method produces greater amounts of high quality plasma DNA at a significant cost savings Using deep well plates inoculate one milliliter per well of luria broth containing appropriate antibiotic with e coli transformed with the desired plasmid DNA culture overnight at 37 degrees Celsius pellet.

The e coli in the culture plate at 5, 000 times gravity for 10 minutes. Then decant or aspirate the supernatant resus bend each pellet in 100 microliters of Resus suspension buffer containing RNAs. A add 100 microliters of lysis buffer per well and mix using a plate shaker for two minutes.

Add 100 microliters of neutralization buffer per well and shake for two minutes. Transfer the cell lysate to the lysate clarification plate. Mount the DNA binding plate on top of the 350 microliter collection plate and place into the bottom of the vacuum manifold.

Now assemble the vacuum manifold as indicated. Place the lysate clarification plate on top of the vacuum apparatus. Apply vacuum at 10 inches of mercury and collect the filtrate into the DNA binding plate.

Do not use a vacuum greater than 12 inches of mercury. Otherwise, fluid will leak through the DNA binding plate. Turn off the vacuum and release pressure from the device, disassemble the vacuum manifold.

Now place the two milliliter waste collection plate in the bottom of the apparatus. Move the DNA binding plate to the top of the manifold. Add 300 microliters of binding buffer per well and pipette up and down to mix.

Apply vacuum at five inches of mercury or greater until the wells are empty. The DNA is now bound to the membrane. Wash the wells with 400 microliters of wash buffer per well.

Apply vacuum, then discard the filtrate. Discarding the filtrate is unnecessary. If using a two milliliter collection plate or filtering directly to waste, repeat the wash once with 400 microliters of wash buffer per well blot the bottom of the filter plates on an absorbent towel to dry.

Apply vacuum once more for five to 10 minutes to ensure the removal of residual alcohol. Block the bottom to ensure removal of ethanol droplets. Add 70 mic releases of elution buffer per well.

If higher DNA concentration is desired, the volume can be reduced to 50 microliters per well, but the average total yield will be lower. Incubate the place at room temperature for one minute. The purified DNA can belu by the vacuum or centrifugation.

Place a clean collection plate that is DNAs and RNAs free into the vacuum manifold. Then mount the DNA binding plate on top of the vacuum manifold and apply vacuum at 15 inches of mercury for one minute until all elucian buffer has passed through the DNA binding plate. Collect the purified DNA for the centrifugation method.

Place the DNA binding plate on top of a clean collection plate and centrifuge at 1000 times gravity for five minutes. To evaluate the plasmid preps. Measure the OD two 60 over two 80 on a spectrophotometer and calculate the concentration of the purified DNA gel.

Electrophoresis of spiking experiments indicate a single clear band of similar intensity from each sample preparation stage and demonstrates little if any DNA loss during the filtration based clarification step. This is reflected in a quantitative analysis of the lysate before and after clarification compared to other similar methods, the highest DNA concentration and total yield has seen from Paul's one milliliter acro prep advanced DNA purification filter plate with 50%more DNA than its nearest competitor. When purified pcat, DNA alluded from acro prep, advanced DNA binding plates using the centrifugation and vacuum methods are compared, the DNA yield is consistently in the supercoil form and with similar concentration.

Now that you've viewed this demonstration, you should be able to easily implement this streamlined method for purifying high yields of quality plasma DNA, and at a significant cost savings.