This protocol provides a technique for increasing the efficiency of 96-well DNA extraction plate loading while simultaneously decreasing the risk of sample cross contamination. This technique reduces the chances for contamination during the critical first step of DNA extraction through the individual addition of samples to each well in the 96-well plate. This methodology can be applied to any of the diverse fields of microbiome research.
Before beginning an experiment, mist the bench top with 70%ethanol. After wiping, let the bench air-dry before spraying the bench with 10%bleach. After wiping and air drying, dip the microscopular, spatular, and curved surgical scissors into 95%ethanol and expose the tools to a flame.
Then dip each tool in 10%bleach and allow the tools to air-dry. Prior to sub-sampling, ethanol sterilize gloves and thoroughly homogenize the soil samples. Next assign a sample ID with well location to each tube.
Fill 95 labeled two milliliter centrifuge tubes with one sample per tube, until each tube is approximately half-full. The 96th tube should be used as an extraction blank. Place the sub-sample tubes on ice, and label 96 sterile 200 microliter flat-caped PCR tubes according to the well labels for a 96-well plate, then place the label 200 microliter tubes in order in a 96-well rack.
To prepare a 96-well plate for the experiment, remove the cover from the plate and place the cover into a sterile plastic bag. Seal the bag to prevent contamination and cover the plate with a pre-cut piece of pierceable sealing film. Then use a rubber roller to firmly adhere the seal to the plate and store the plate at four degrees Celsius.
To transfer the sub-samples to the plate, place 24 of the two milliliter sub-sample tubes into an ice block for cold storage, and use the sample name and well location sheet to select the appropriate corresponding 200 microliter flat-capped tubes. Vortex the sub-samples one at a time, for five seconds per sample to ensure homogenization and load approximately 200 microliters of each sample into the appropriate corresponding PCR tube. When all 24 of the samples have been loaded, use a bleached soaked paper wipe to clean the outside of one PCR tube, and invert and tap the tube on the bench to move the sample to the top of the tube.
Using the flame sterilized and bleached scissors clip the bottom of the PCR tube to create an opening for the sample, and pass the tube over the plate with the cut end facing up until the appropriate well has been reached. Tilt the plate slightly, to facilitate puncturing of the pre-cut pierceable sealing film, and with the tube directly above the correct well, quickly but carefully invert the plate so that the cut tip fits into the well. Use a sterilized tool to tap the top of the tube until all of the soil has fallen from the tube into the well.
And leave the tube in the well with the lead closed. When all of the samples have been loaded in the same manner, remove one 200 microliter flat-capped PCR tube and add 750 microliters of bead solution to the sample well. Place the 200 microliter flat-capped PCR tube back into the well pushing it all the way down into the well.
And use a Sharpie to mark the top of the tube to indicate the well has been loaded. When all of the wells have been loaded with beads return the 24 sample tubes to 20 degrees Celsius storage and add 24 new sub-sample tubes to the ice block, to allow the next set of samples to be loaded into the 96-well plate as just demonstrated. When all 95 of the wells have been loaded with sample and bead solution, add bead solution only to the 96th well.
Remove all the tubes, passing them over covered wells only and carefully remove the pierceable film from the plate. Then carefully transfer the plate cover from the plastic bag to the plate and place the plate at 20 degrees Celsius until the planned extraction. A comparison of plate loading methods shows that the demonstrated method results in the lowest DNA concentration within the blank wells.
Using this method, the DNA concentration was significantly lower then when using the method proposed by McPherson et al. Although the DNA concentration by this method were not statistically different than the qiagen default method. All three methods, produce mean DNA concentrations under two nanograms per microliter.
Although only this new method produces wells with no measurable DNA concentration. To minimize the potential for spillage, keep the 200 microliter tube upright when moving it over the plate, tilt the plate and insert the tube into the correct well.
