Automated Sample Prep & Plate Pipetting
Automated Sample Preparation in the CGC
The Center's Qiagen BioRobot 8000 can process up to four 96-well plates (384 samples) under many DNA sample preparation protocols. The plant and tissue genomic DNA prep protocols use a large number of tips and plates and so only 2 96-well plates worth of samples can be processed at a time. The robot is designed to work with Qiagen kits, but it has a very flexible computer control interface that makes it possible to program it to run most any kind of procedure you could create for the microtitre plate format. The BioRobot was upgraded in September 2006 to be able to carry out precision pipetting of small volumes, down to 1uL. This makes it possible to use the BioRobot to set up small volume reactions in 96 or 384 well plates, make accurate dilutions of samples based on plate assay data, and so forth. The 4 precision pipetting probes have a total volume capacity of 100 uL. 300 uL and 10 uL tips are stocked for these low volume pipetting needs.
Users are expected to supply their own Qiagen kit or equivalent, or purchase a kit from the CGC stockroom along with the tips needed for the protocol, and bring a bottle of alcohol for preparing the buffers. Users must supply their own filter plates, buffers, and so on for a custom protocol. Consult the kit handbook to make a list of the necessary supplies. Protocol handbooks for all Qiagen kits can be downloaded from the Qiagen web page in .pdf format. The Robot uses black conductive Qiagen tips and plain clear tips for pipetting. Users accounts will be charged for the time running the machine. An initial training session is required for new users of the BioRobot. The user's account will be charged $40.00 for the initial training, after which the normal usage charges apply. BioRobot usage for sample preparation is charged by the hour. The center Director can also create, or help you create, custom protocols for specific projects at the current staff time recharge rate.
A clear plastic cover with an integrated HEPA filtration unit was built for the working deck of the BioRobot in 2007. This makes it possible to handle and transfer cultures with minimal risk of contamination. This feature may also have other applications, such as in critical PCR plate set ups and sample preps.
The Apricot Personal Pipettor can do sample transfers, dilutions, etc., in 96 or 384 well formats using either low volume teflon-coated stainless steel fixed tips, or heads that use Matrix disposable plain of filtered tips with volumes ranging from 12 uL to 500 uL.
The Qiasoft operating software will direct the user in how to prepare the Robot for a sample preparation procedure once the user has selected the protocol they will use from a list, and input the number of samples to be processed. Starting a protocol and working through the set-up messages is the best way to find out how many racks of tips and other supplies will be needed, so it is a good idea to make a "dry run" of your intended protocol before ordering supplies.
It is also worth checking the BioRobot section of the protocols archive page to see if another user may have found a better or less expensive way to do the kind of sample processing you have in mind. The center posts useful protocols and user tips on that page when a user finds a method or experimental design they think is especially good.
To avoid cross-contamination problems during downstream PCR procedures, filter tips should be used for any genomic DNA preparation protocols. Unfiltered tips are acceptable for plasmid DNA preps and PCR product purifications.
A booklet containing sign-up sheets for Robot usage and for charging tips and kits to your budget number is kept next to the Robot. The price list for the tips and kits is inside the cover. Users can fill out the charge sheet for tips and Elution Microtube RS plates for collecting the processed samples once they are finished setting up the Robot deck and know how much of what is actually going to be used.
The CGC currently has pre-programmed protocols which run the following
Qiagen kit methods:
1.) QiaQuick PCR reaction clean up.
2.) R.E.A.L Prep rapid plasmid DNA prep, from 1.5 ml O/N cultures.
3.) QiaQuick Turbo plasmid mini-prep, from 1.5 ml O/N cultures.
4.) DNeasy Tissue genomic DNA prep.
5.) DNeasy Plant genomic DNA prep.
6.) Eppendorf PerfectPrep 96 plasmid DNA prep.
The CGC has also programmed a few generic protocols for preparing
reactions and samples in microplates:
1.) Set up PCR reactions in 96 or 384 well plates from user-supplied input tables which specify the name, position, and concentration of templates, and the pattern for up to 8 different master mixes.
2.) Standardized sample dilution, which prepares a plate where the concentrations of all the templates is the same based on a user-supplied table containing concentration data. This allows templates to be distributed from the standardized plate into a reaction plate all at once on the Apricot Personal Pipettor using the washable steel tips. This gets the job done more quickly and saves the expense of all those disposable tips needed to transfer template samples.
The Apricot Personal Pipettor is run by a simple computer interface which allows the user to program a sequence of actions involving aspirating and dispensing liquid volumes from the 4 sample stations on the plate deck, and automatically washing probes or tips. Aspiration can be from plates or from troughs arranged for row or column pipetting. The overview section below explains how it works. The user only needs to know what volumes they want to transfer and decide in which positions to place their plate(s). If you are using an unusual type of plate you may have to "teach" the plate. Ask the lab director for assistance with this task.
An initial training session is required for new users of the Apricot. The users's account will be charged $40.00 for the initial training, after which the normal per plate charges apply to normal usage. Users will have to buy their own racks of Matrix tips for the disposable tip heads. Programming and protocol development and testing time is charged at $10.00 per hour.
Above Pipetting probes picking up eight 1100 ul tips.
Lab hand carrying dispenser head over a 96-well plate on the vacuum manifold, delivering buffer to rows of eight wells.
Lab hand lifting QiaPlate off of the vacuum manifold.
Below Pipetting into a 96-well plate on the vacuum manifold.
The BioRobot has three primary tools it uses to handle samples and liquids, the 8 pipetting probes, the lab hand, and the 8-channel dispenser head. The pipetting probes can work individually or in groups of up to eight at a time. With the conductive tips the probes can sense the level of buffer in a reservoir and the robot can then calculate whether the user has supplied enough buffer to process the intended number of samples. The lab hand can lift and move microplates, vacuum manifold parts, adaptors, lids, etc. The lab hand is also used to distribute buffers from a bottle selected from the rotating rack by picking up the dispenser head and carrying it over a plate. These functions are illustrated in the adjacent pictures.
The robot keeps track of the positions of the various samples, adaptors, and parts, by alway placing these items in one of the 26 defined postions, or "slots", on the deck. The arrangement of the deck, or work table, can be seen in the lower frame in the QiaSoft screen shot, below. The BioRobot deck has stations where vacuum filtration or drying can take place and where heating or shaking can be done. It has the ability to pipette liquids from up to 5 different reservoirs which each hold up to 80 ml, and to dispense buffers to 8 plate wells at a time (one row on a 96-well plate) from any of 6 bottles with 250 ml or 500 ml capacity, in a rotating rack. The pipetting function can make simple transfers using 1 to 8 pipette tips, and it can mix the contents of a well by drawing a volume up and down several times in the same tip.
Apricot PP125N-XD is an automated plate pipetting station with exchangeable heads and a four postion plate deck. The deck can move both left to right and front to back. If you use one row or one column of disposable tips this allows dilutions or transfers by row or by column, in addition to full plate at once transfers. This system includes a computer controlled pump which allows washing steps to be carried out automatically as part of a pipetting program.
In this picture there are 3 plates in deck postions 1, 2, and 3, and the plate washing station in position 4. The frames below outline an example of how a pipetting program is created using these plates and accessories. At the end of this presentation you can watch a short low-res video of the program running.
The Apricot is operated by PC software which has a simple graphical user interface. The overall procedure is to select a pipetting head (screen shown at right), select plates from the list of "taught" plates available and place them into one of the four deck postions, then create aspirate dispense and wash steps as desired.
Each pipetting head has it's part number stamped on the front. The 125uL 96 well fixed tip head is shown installed in this picture. It is the head choice highlighted in blue in the previous picture.
The head selection screen. Only a few example heads were "taught" at the time these pictures were taken. Two of them are wash stations that work with a computer-controlled peristaltic pump. the masterFlex pump is noted as being ready in the lower right corner of the screen.
An example aspirate step. Deck postion #1 is chosen, where 20uL will be picked up. The tips will not be placed at the bottom of the well, but at about half depth. This assumes that the wells be at least half full of liquid.
Dispense steps work the same way, and Wash steps are also very similar.
Here is a full screen showing a simple completed program that will pick up 20 uL from the plate in position #1 and dispense it into position #2, then wash the head 3 times at postion #4 using the MasterFlex pump to provide continuous fresh wash water, and dispense any remaining liquid at postion #3.
Close up of the example
It's just that easy!
The link at right is for a PowerPoint slide show which demonstrates two different ways to use teh Apricot.
The first part demonstrates the use of the 384 Stainless tip head to transfer 10uL from one plate to another. The second part demonstrates the use of the 384 disposable tip head to dispense 1 ul from a column of reservoir wells in one plate to all positions in another. The nice feature of this is that there is essentially no waste of the reagent being dispensed.
You can watch the example program shown above run. Click on the Apricot video link at right.