The overall goal of the following experiment is to isolate human genomic, DNA from whole blood, using true tip extraction technology with the Hamilton Star Liquid handling workstation. To achieve this lys cells in solution comprised of the blood sample lysis buffer proteinase K and ethanol are aspirated and dispensed through the true tips multiple times, thus binding the DNA to the true tip matrix. Next two separate wash buffers are flowed through the true tips in order to remove residual proteins, lysis, buffer, and other contaminants from the bound nucleic acids.
In the third step, the binding matrix of the true tip is dried by aspirating and dispensing air multiple times to evaporate any remaining wash buffer in the final step, illusion buffer is flowed back and forth across the binding matrix to release DNA from the matrix. Using true tip technology contaminant free high molecular weight Genomic DNA is thus extracted and can be confirmed by UV vis absorbance, gel electrophoresis and quantitative PCR or sequencing. The advantages of true tip over other sample preparation methods based on spin columns or magnetic particles is that Tru Tip is easy to automate.
It does not require costly systems with centrifuges and magnetic rods, and the simple workflow saves valuable deck space. Though we'll focus on whole blood today. Tru Tip can be applied to other genomic DNA sample applications as well as viral RNA from clinical swab samples or the isolation of cell-free DNA from plasma to name a few Demonstrating the procedure will be Dane Brady, an automation engineer from our laboratory who has been instrumental in the development of the Truet tip extraction procedure on the Hamilton Liquid Handling Workstation.
In this protocol, a Hamilton Star Liquid handling robot simultaneously extracts DNA from 96 wells of whole blood. The Hamilton Star has an optional heater shaker unit on the deck, which is important for enzymatic digestion of certain clinical matrices such as Whole blood. Because the system can be fitted with a 96 channel pipette head, there is a dedicated 96 well plate for each of the true tip steps and reagents.
First turn on the Hamilton Star robot associated equipment and the computer on the computer. Open the Hamilton Run Control software in the software. Open the run file provided by ACON for 96 samples.
Now set up the star deck layout following the diagram. Place one milliliter filtered tips in all the positions labeled one in position two place unfiltered, one milliliter tips in Position three, place a Conni Hamilton one milliliter LPT two millimeter true tips. Continue referencing the chart to fill the remaining positions from five to 12 with the required LabWare, but do not load the samples yet.
Now dispense the reagents 75 milliliters of lysis and binding buffer. F should be loaded in trough position five. Alternatively, plates prefilled with buffers could also be used to streamline the setup and simplify the deck layout.
Continue loading the robot accordingly. Lastly, remove the caps from the blood tubes and place them in the sample carrier racks or deck position four. Place sample one in the rear of the far left carrier and move sequentially down each carrier with sample 96 ending in the front right position.
Once all the samples are loaded, allow them to equilibrate to room temperature. Because the protocol is completely automated, there's little opportunity for operator error. The samples need to be placed in the correct order in the rack, but bar coding can be implemented to ensure proper sample tracking.
To start an automated script, select the play button in the upper left corner of the run file window and input the number of samples being processed. The first step executed by the script is to transfer 80 microliters of proteinase K to the incubation plate at position 14 on the heater shaker module. Next, the script dispenses 200 microliters of sample into each sample well of the incubation plate using the eight channel arm.
This is followed by dispensing 600 microliters of lysis buffer F into each well of the incubation plate using the 96 channel arm. The script then mixes the solution for 10 cycles and incubates them for 20 minutes at 70 degrees Celsius and 500 RPM in the deep well plate, the temperature is only 60 Celsius, which is within the range of proteinase K activity. While the samples are incubating, the liquid handling system continues operating by dispensing the extraction and elution reagents from the bulk reagent troughs to their corresponding 96 well plates, 100 microliters of Elucian buffer A is added to each well of the deep well plate.
At position 13, 800 microliters of ethanol is deposited into each well of the deep well plate. At position ten one point six milliliters of wash buffer K is added to each well of the deep well plate at position 12 and 1.6 milliliters of wash buffer J is added to each well of the deep well plate at position 11. After the 20 minute incubation, the sample mixture is transferred from the incubation plate to the deep well plate containing ethanol at position 10, where it is mixed by 12 pipetting cycles.
Throughout the process, reagent tips are rejected into the waste bin as required. The program continues from the lysis process to the DNA extraction by first loading 96 true tips from deck position three. The next step is to aspirate and dispense the samples in position 10 over 10 cycles, thus binding the nucleic acids to the true tip monolith.
Next, the true tips move to position 11 at which wash Buffer J is cycled over the monolith five times to remove residual lysis, buffer, and sample matrix. Then the true tips move to position 12 where wash buffer K cycles through them five times to remove proteins and other contaminants from the bound nucleic acids. Next, the true tips air dry by cycling 80 times at high speed above the wash plate.
Now, the true tips move to position 13 where elucian buffer a two cycles through them five times releasing the DNA into the deep well plate. The true tips are now ejected into the waist bin. Now the program is finished.
Remove the elution plate from the instrument and transfer the extracted samples to the appropriate tubes for storage or downstream applications. The program can also be customized to automatically transfer the final samples to the desired tube or plate of choice in just one hour. The automated system extracted genomic DNA from 96 blood samples using the robot.
The absorbance profiles of 45 positive blood samples was processed simultaneously with 45 reagent blanks. An average two 60 to two 80 absorbance ratio was 1.96, and the average two 60 to two 30 ratio was 1.93 suggesting pure samples on a 1%aros gel. Random samples ran at a high molecular weight with minimal shearing.
The average yield was about five micrograms of DNA per 200 microliters of whole blood. When optimizing the automated true tip protocol special care is taken to make sure there is no cross contamination between wells. Real-time PCR shows clean separation between positives and negatives as shown here.
For RNA extractions, The entire extraction of 96 samples can be completed in under one hour with minimal hands-on time required. After watching this video, you should have a good understanding of how to isolate human genomic DNA from whole blood using the TRU Technology Automated on the Hamilton Star Liquid Handling Workstation.
