Method: Restriction Digests of Human Genomic DNA Using the Biomek 1000 Workstation

Srini Ramachandra

The Beckman Biomek 1000 is an automated laboratory workstation designed to carry out rapid and accurate liquid handling operations such as pipetting, diluting and dispensing liquids. In our lab the Biomek is mainly used for doing human DNA restriction digests in a 96-well microtiter plate format. The restriction digests are done under four main functions namely: enzyme-cocktail delivery, DNA delivery, lambda DNA delivery, and setting up test digests. A brief description of the workstation and the various methods & subroutines is given below. Refer to the Biomek 1000 manual for more information on writing and building subroutines and methods.

Time required:

approximately 2 hours for 96 samples

Special supplies required:

V-bottom sterile microtiter plates (Dynatech), sterile disposable caps for the microtiter plates (Beckman, Cat.# 267002), sterile P-250 tip rack assembly (Beckman, Cat.#372655), 17 ml modular reservoir divided by length (Beckman, Cat.#373692).

Description of the workstation:

The Biomek consists of three parts: the workstation with a robot arm, the electronic interface unit (EIU) and an IBM PS/2 Model 50 computer. The workstation is run by the EIU which interprets commands from the Genesis software (version 2.01) installed on the PS/2.

Description of methods :

All commands are built into a set of subroutines and methods. Running the workstation deals only with the methods. The following methods use both the single-tip and the multi-tip pipetting tools and are designed for individual digest volumes for 2 gel lanes.

Precautions:

The tip racks, microtiter plates and the microcentrifuge tube holders should all be seated properly between the steel guides on the trays. If they are not seated properly the tips will be dragged over breaking the tips and damaging the tools in the process. The person operating the Biomek should watch all operations closely and catch any errors in delivery of solutions or malfunctions of the Biomek. Do not leave the workstation unattended for any length of time. The pipetting tools are very expensive and therefore require a lot of caution and attention.

Procedure:

Day 1

1. Take the CEPH DNA tubes out of the cold room for the particular set of families you are working with. Flip open the lids of the eppendorf tubes, place them in the Biomek microcentrifuge tube racks (with white inserts) starting with the first individual of the first family. Bend the lids back and tuck them in the space between the white inserts and the rack. Each rack can hold 24 tubes. All families for any one method can be accomodated in 4 racks. See figure below for order of samples:
   

2. Cover all the tubes with parafilm (sterile side down) stretched over them and warm up the DNA in the 50 degrees C incubator for 30 minutes. This helps reduce the viscosity of the DNA.
3. Meanwhile label two microtiter plates with the following information: Method, enzyme, date, your initials and family I.D.#s. Label one plate as MAIN DIGEST and the other one as TEST DIGEST.

4. Using the enzyme-cocktail calculation sheet work out the amounts of enzyme, 10X restriction buffer and sterile water required. Always make up an excess of the enzyme cocktail to make up for pipetting losses. For example, for 85 individuals prepare enough cocktail for at least 100 individuals. (Refer to the table at the end of this procedure for enzyme-cocktail calculations).
5. Place a new box of tips in the TIPS tray, the 17 ml reservoir holder in TRAY 1 and the MAIN DIGEST plate in TRAY 2 as shown below:
   

6. Prepare the enzyme-cocktail and pipet it into the left half of the 17 ml modular reservoir. Place the reservoir in the left quarter of the holder.
7. Turn on the PS/2 monitor, the computer and the EIU. The robot arm will go through some priming movements. When the Biomek Main Menu appears on the screen select 1 to access RUN METHOD. Pick a method from the list of methods on the screen and press ENTER.
8. A message will remind you to turn the vacuum on. There is no vacuum unit for this Biomek, so as a default press ENTER.
9. A series of messages will take you through the configuration of the pipetting tools and the tablet. Double check the configuration against what you have on the tablet and press ENTER each time.
10. The Biomek will dispense 18 µl of enzyme cocktail to the MAIN DIGEST plate using a combination of the MP20 and the P20 tools.
11. After the enzyme-cocktail is dispensed a pause-till-continue function is executed. The Biomek will pause for the next command. Place the first two sets of DNA tubes in TRAYS 1 & 3 respectively as shown below:
    

    Press ENTER and the Biomek will start dispensing 36 µl of DNA from each tube to its respective well using the P200 tool.

12. After the first two trays are dispensed a message appears on the screen and another pause-till-continue function is executed. Replace TRAYS 1& 3 with the next set of tubes. Press ENTER. 36 µl of DNA is again delivered to the microtiter plate starting after the last well that the DNA was delivered to in step #11.
13. Place a 17 ml modular reservoir containing at least 3 ml of 500 ng/µl lambda DNA in TRAY 1 as described in step #6. A 17 ml reservoir labelled "Lambda DNA" is always kept in the refrigerator covered with parafilm and can be reused. Cover the reservoir with fresh parafilm after each use and store the lambda DNA at 4 degrees C. Place the microtiter plate labelled TEST DIGEST in TRAY 3 and press ENTER. The Biomek will deliver 2 µl of lambda DNA to each well of the TEST DIGEST plate using a combination of MP20 & P20 tools.
    

14. The Biomek will continue without any breaks and dispense 6 µl of the digest mix from the MAIN DIGEST plate to the corresponding well in the TEST DIGEST plate. Except for enzyme cocktail and lambda DNA delivery all other delivery functions include 3 MIX cycles and a TIP TOUCH function. Manually set up a lambda-only digest control in one of the empty wells or an eppendorf tube.
15. While the Biomek is preparing the test digests, warm up the disposable caps in the 37 degrees C incubator to soften the caps. This will enable you to place the caps easily over the microtiter plate. At the end of the method, press ENTER and the computer will take you back to the Main Menu. Select 5 to exit to DOS, press ENTER and turn off the monitor, the computer and the EIU.
16. Spin the plates in the microtiter plate holders in a Beckman J-6 centrifuge at 2500 rpm for 2 minutes to bring down the drops of solution left behind by the TIP TOUCH function on the top edge of the wells. Place the disposable caps tightly on both the microtiter plates, wrap the edges with parafilm and place the plates at the appropriate temperature for the enzymeincubation. If incubating above room temperature, place a wood block on the plates and a 500 ml bottle filled with water on top of the wood block as a weight to keep the caps from popping open when they expand due to the heat. Incubate the digests overnight.

Day 2

1. Store the MAIN DIGEST plate at -20 degrees C. Add 10 µl of 2X Ficoll dye to all samples in the TEST DIGEST plate, load them on a double-combed 0.8% TA-agarose gels (20cm x 20cm) along with the lambda digest control and 1kb ladder marker and carry out electrophoresis at 80V for approximately 2 hours. Compare the lambda DNA bands in the human DNA digests against that of the lambda control digest. The partial digests usually will have bands missing while showing a few extra high molecular weight bands. The uncut digests will have a bulk of the DNA very close to the wells with no clear bands showing.
2. Depending upon the results of the test digest, add 20 µl of 10X Stop dye to the digests (MAIN DIGEST plate) that are complete, add 4-5 units more enzyme per µg of DNA to the ones that are incomplete digests and incubate the entire plate overnight as in step #16.

Day 3

1. Run another test gel on the samples that were incomplete. Based on the test gel results add 20 µl of 10X Stop dye to the samples that are complete. If samples are still incomplete, prepare new digests manually. After all the digests are complete, store the MAIN DIGEST plate (with 10X stop dye in all the wells) at -20 degrees C.
2. Fill out the digest template sheets and the test gel sheets. The digest template sheets indicate loading order of the samples, the family I.D. #s of that particular digest plate and the volume of digest left in the wells. The test gel sheets will tell a person whether a digest was complete or not and if there is any problem with the status of the DNA in question. File them in their respective folders which are kept behind the Biomek on the shelves.
3. The method can be temporarily stopped anytime during the run by pressing the /STOP or /PAUSE keys. These "soft" keys can be accessed by pressing the / (slash) key. The run can be continued by pressing the /CONTIN soft key. If the Biomek malfunctions, press the red EMERGENCY STOP lever in front of the workstation. This will stop the Biomek immediately and will abort the method currently running. Once a method is aborted you will have to start a new method and cannot continue with the aborted method.

Enzyme-Cocktail Calculations: