Appendix: User's Guide to the ABI Nucleic Acid Extractor

June 26, 1990

Matthew Holt

Description

The Applied Biosystems Inc. 340-A Nucleic Acid Extractor is an automated instrument-reagent system for the purification of nucleic acids from viruses, cells, tissues, and body fluids (although all we have used thus far is human cells). With the 30 ml vessels, the extractor has the capacity of extracting DNA from up to 2 x 10e8 cells.

The 340-A Extractor programming, version 2.11-30 ml, has its operations stored in three "MAIN MENUS", only one of which will appear on the screen at a time. One can scroll through these menus by pressing the "Main Menu" key. The operations are as follows:

MENU 1

• RUN CONTROL
• RUN MONITOR
• REVIEW & EDIT METHODS
• MANUAL CONTROL

MENU 2

• PURGE
• SELF TEST
• BOTTLE STATUS
• PRINTER

MENU 3

• POWER FAIL
• SET CLOCK

The programming for cell extraction is currently in "Method 1", and the programming for the once-a-month purging, "SHUT", is in "Method 2". "Method 1" employs 5 "procedures" (called P1, P2, P3, P4, P5). Method 2 consists of 2 procedures (P6, P5). NOTE: a Method is an ordered list of procedures, abbreviated by a capital "P" when locked, or a small "up" when unlocked to allow an edit. A procedure is an ordered list of functions. A function is equivalent to one step for the extractor, or one set of instructions for one particular event and usually requires up to four parameters. Functions are numbered 1-73, each number representing a unique operation. See section 4.1.4 of the users manual for the list of functions. Be careful not to confuse function numbers with step numbers (which only tell you where you are in a certain procedure). See below for a description of each procedure currently in use:

P1 - DIGEST: This is copied from the EPROM memory cartridge program "DDig" with a few modifications:

• Vessel size- all volumes have been adjusted to accommodate 30 ml vessels.
• Digest time is increased to two hours.
• Rocker speed is increased from 8 to 4 (NOTE: 1 is fastest speed) to ensure proteinase K distribution.
• R4 delivery of proteinase K is written out because of line clogging problems. Hence, proteinase K is delivered by hand immediately after loading sample at Step 19.

P2 - PHENOL/dH2O/CHLOROFORM EXTRACTION: This procedure is copied from the EPROM cartridge program "DExt1". (NOTE: Method 1 has two P2 in its list because the extractor executes this procedure twice.) The modifications are as follows:

• Vessel size- all volumes adjusted for 30 ml vessels.
• Mixing time- mixing time is increased to 20 minutes.
• Speed- speed increased to 3.
• Educe function- All rapid educe functions have been replaced with slow educe functions (#66). The parameters have also been adjusted to allow the extractor to continue educing for two seconds after the interface has been detected to minimize debris in sample. Maximum detection time is set at 120 seconds, minimum detection time set at zero seconds.

P3 - CHLOROFORM: This procedure is copied from the EPROM cartridge program "DExt2" with the following adjustments:

• Vessel size- All amounts adjusted for 30 ml vessels.
• Speed- Speed is increased to 3.
• Educe- Rapid educe functions have been replaced with slow educe functions, along with their corresponding parameters, just as in "P2".

P4 - PRECIPITATION: This procedure is copied from EPROM cartidge procedure "DPpt1" utilizing isopropanol ("DPpt2" uses EtOH). The changes are as follows:

• Vessel size- All amounts adjusted for 30 ml. vessels.
• 3M NaAcetate delivery - NaAcetate is currently routed through R5 (reagent port 5) in P4 at step 9 for 9 seconds for each vessel (approximately 2 ml). R5 previously contained 100 mM Tris buffer used in proteinase K delivery which has become obsolete because of the elimination of automated proteinase K delivery.
• Shake Rocker- Mixing time is doubled and speed reduced from 1 to 3 in an attempt to be as gentle as possible.
• User Interrupt- At step 22, the procedure calls for the use of "precipitettes", but we do not use them because of problems with the DNA sticking to these "precipitettes". At step 22, the menu will read "USER INTERRUPT". The vessels will be inverted to prevent heavy pellets from being drawn into the drain. (The extractor delivers reagents form the top and drains from the bottom of the vessels.) At this point the operator must retrieve the DNA pellets. The rocker must first be postioned upright.To do this, press "MAIN MENU" key until Menu 1 appears, find and press "MANUAL CONTROL" followed by "ROCKER" and then "DOWN". Hold "DOWN" until the rocker is upright. Open top of vessel and pull DNA pellet with a sterile transfer pipet. After the DNA has been removed, continue the procedure by pressing "MAIN MENU", then "RUN CONTROL" followed by "RESUME RUN" and then "RESUME STEP". The remaining five steps in "P4" prepare the extractor for a cleaning purge EPUR, "P5".

P5 - EPUR: This procedure is copied from the hard disk procedure EPUR (extensive purging) with vessel size indicated as 30 ml.

P6 - SHUT: The SHUT purge (formerly called TST5) is copied directly from the hard disk procedure SHUT with vessel size indicated as 30 ml. ("Method 2 consists of P6, P5). Always follow a SHUT purge (P6) with an extensive purge (P5).

Operation

1. Turn the extractor on by pushing the black toggle switch on the left of the front of the machine. Once the machine is on, the menu will appear and say " MAIN MENU TO BEGIN". Push MAIN MENU, then RUN CONTROL, and in RUN CONTROL is the message SET-UP RUN. Push SET-UP RUN and the message will read:

   METHOD 1 VESSEL SIZE: 30 ML.

   P1 P2 P2 P3 P4 P5 ACTIVE VESSELS: 1 2 3 4 5 6 7 8

   The underlines indicate where to place the cursor to change something. For instance, to execute Method 2 (the SHUT purge) place the cursor on "1" and press PREV or NEXT keys to scroll until 2 appears on the screen. Be sure that all vessels are listed in active status. For example, ACTIVE VESSELS: 1 2 - 4 5 6 7 8 shows that vessel 3 has been deleted. Reinstate vessel 3 by placing the cursor on the "-" and push PREV key. Move the cursor by pressing arrows on the keyboard on front of the machine. Note: all current procedures for cell extraction are in "Method 1".

2. Check the bottle status: Before starting a run you MUST review reagent status and replace any low bottles or dump the waste bottles. To do this, flip the toggle switch on the right side of the machine from "pressurize" to "vent". Bottles should be replaced when they are 2/3 empty. The remaining reagents can be added back into the system at a later time. Never use a bottle that is full, for the machine uses helium gas to pressurize the bottles and needs some empty room in each bottle. The reagents are as follows:
   • R1- nothing (formerly 3M NaAcetate, 0.2 micron filtered)
   • R2- nothing
   • R3- nothing
   • R4- nothing (formerly proteinase K - Because of line clogging problems, proteinase K is delivered by hand at the time of sample loading. Use only ABI proteinase K (Cat #400457). It comes lyophilized in a 15 ml brown bottle in an amount defined by the particular activity of each lot of proteinase K, so one can always use this recipe to resuspend the enzyme: To one unopened bottle add 12 ml 0.2 micron filtered dH20 + 240 ul 1M Tris pH 8.0, 0.2 micron filtered. Use one ml per vessel per run. Lyophilized stability is one year at -20 degrees C. When resuspended it can be stored at room temperature for one week, at 4 degrees C for two weeks, or longer at -20 degrees C but should not be thawed more than once.
   • R5- 3M NaAcetate pH 5.2, 0.2 micron filtered (formerly 100 mM Tris pH 8.0, 0.2 micron filtered)
   • R6- IN Nitric Acid, 0.2 micron filtered
   • R7- Chloroform ASC grade
   • R8- Phenol/dH20/Chloroform, purchased from ABI Cat #400765
   • R9- 2X Lysis Buffer, purchased from ABI Cat #400676
   • R10-dH20, 0.2 micron filtered
   • R11-95% ETOH, UPS grade, 0.2 micron filtered
   • R12-Isopropanol, UPS grade, 0.2 micron filtered

   AQUEOUS WASTE

   ORGANIC WASTE - with Phenol, Chloroform and water

   HELIUM GAS - purchased from Cee-Kay, #29, dual regulator set at 60 PSI,

   one tank lasts 2-3 months depending on use

   Once you have replaced a reagent you must pass this information on to the extractor. Find and push BOTTLE STATUS on MAIN MENU 2 which then reveals two options: REVIEW BOTTLE STATUS and PRESSURIZE BOTTLES. Press REVIEW, and the extractor will ask how much of each reagent you have. It uses this information to trigger its alarm when a reagent is low by subtracting from this amount when it uses a reagent. The extractor cannot sense an empty bottle or the lack of flow of a reagent. NOTE: if a reagent bottle is empty and the extractor memory states a volume for that reagent, nothing will be delivered to the vessel and no alarms will be triggered. Also NOTE: the extractor does not keep track of the waste it generates, so you MUST review the status of the aqueous and organic waste bottles before each run. These bottles should be emptied when they reach 2/3 capacity. Aqueous waste can be disposed of down the drain provided the nitric acid has been neutralized with Na2HPO4. When this bottle is emptied, therefore, you must add 100 g Na2HPO4 . The organic waste must be documented and removed by Washington University Hazardous Waste Management Office, Box 8229, 362-6816. After REVIEW BOTTLE STATUS is completed, you are ready to pressurize the system by pushing the PRESSURIZE BOTTLES, then press ALL for all reagent and waste bottles. It takes approximately 4 minutes for the extractor to pressurize the system.

3. Run a SELF TEST: Once all reagent bottles are pressurized you can have the extractor perform its SELF-TEST by scrolling to MAIN MENU 2 and pressing SELF-TEST, then ALL TESTS to perform all test functions. The SELF-TEST program takes about one minute to complete. The extractor will print a message "ALL TESTS OK", and you are ready to start the run.

4. Start the run: Return to MAIN MENU 1 and push RUN CONTROL, then START RUN, then START. The first step in the program is "INSTALL RUN CONNECTORS", so after pressing START you will hear an alarm and see the message to install run connectors (NOTE: the extractor will make this noise any time there is an event that needs user intervention to continue). Place "fat" end of connector in bottom of vessel securely and close top and bottom clamps. Once all connectors are in place, press RESUME to continue. The next steps of the program check these connectors for leaks. If there are no leaks, the machine will continue on with the run. If it detects a leak you must:
   1. press INTERRUPT.

   2. determine which vessel leaked by checking SET-UP RUN (in MAIN MENU 1

      under RUN CONTROL) to see which vessel(s) was deleted. NOTE: you must reinsert the deleted vessel or the machine will ignore it for the rest of the run.

   3. Tighten or replace that run connector.

   4. RESUME run at Step 2, function 46, at the vessel in which the leak was first detected so that another leak test can be performed on that vessel.

   5. Load the samples: Approximately 10 minutes after the leak test is completed, the extractor will sound its alarm and print the message "LOAD SAMPLE". The cells, resuspended to a total volume of 4.5 ml in 0.9% NaCl, can now be added directly to each vessel. NOTE: you must be careful not to load cells on front wall of the vessel because they will not get mixed properly. After cells have been added, then add 1 ml of proteinase K to each vessel, again avoiding the front wall, close the vessels, and press RESUME.

   6. After two hours of proteinase K digestion, the extractor executes two phenol/dH2O/chloroform extractions and one chloroform extraction. The extractor adds and mixes the reagents and then allows the phases to separate before educing the lower phase. Note: it is a good idea to make sure each vessel interface is clearly defined prior to the educing steps. The extractor has a maximum time of 2 minutes to detect the interface in a vessel. Usually this is quite adequate to empty all of the lower phase, but occasionally it will need a little more time and its alarm is triggered and it prints the message "AQUEOUS PHASE NOT DETECTED". If you do not catch this problem immediately the extractor will continue to educe all vessels and then stop all activities. In this case you would have one or more vessels that still have the lower phase to be educed.

   To correct this you must:

   1. press INTERRUPT.
   2. MAIN MENU 1, RUN CONTROL, SET-UP RUN, and delete all vessels except those that still need educing.
   3. MAIN MENU 1, MANUAL CONTROL, FUNCTIONS, then enter function number 66 EDUCE with 4 parameters in this order: 2 (additional draining for 2 seconds after interface is detected) 6 (helium cycling time of 6 seconds) 0 (minimum detection time) and 120 (maximum detection time). NOTE: when in manual control, the extractor will only execute the function you enter and it will stop after that function is completed. Also NOTE: when in manual control doing a function that generates waste, you must first enter the function number to direct the waste to the proper bottle. In this particular case the organic waste bottle has already been selected for educing.
   4. MAIN MENU 1, RUN CONTROL, SET-UP RUN, and reinsert all vessels.
   5. MAIN MENU 1, RUN CONTROL, RESUME RUN, either RE-DO STEP, NEXT STEP or JUMP, depending upon how the run was interrupted.

      Occasionally, for viscous samples, the extractor will think it has educed all the lower phase of one vessel. It may have detected a small amount of the aqueous phase and moved to the next vessel. In this case, no message will be printed on the screen and the extractor will continue the run. This could be a problem if subsequent steps add too much volume for the vessel. (Hence, again, it is important to verify that the first extraction interface is well established and the first educing goes without problems.) Correct this just as you would if it printed the message "PHASE NOT DETECTED". Sometimes (very seldomly) the extractor will attempt to educe the vessels, but because of the high viscosity of the aqueous phase it actually pulls some of the aqueous phase through the lower organic phase. In this case you have lost your sample and should delete this vessel from the rest of the run to save reagents. Be sure, however, to reinsert this vessel before running EPUR so that the vessel gets cleaned.

   6. P4 step 22 is a USER INTERRUPT approximately 4 hours after the start of digestion. At this point the DNA pellets should be floating free in the vessel. If there appears to be an "anchor" of clear DNA which causes the pellet to sink, add more 3M NaAcetate and re-mix. By hand add 500 ul and to mix, go to MAIN MENU, MANUAL CONTROL, FUNCTIONS, 61 SHAKE ROCKER with 4 parameters in this order: 120 (time in seconds), 140 (angle of rocker), 3 (speed), and 0 (delay in seconds). When the DNA pellets are floating free, gently remove them with a transfer pipet (we have extra long ones especially for the extractor), rinse in 6 ml of filter sterilized 70% EtOH, and place the precipitate in 1-2 ml of 1X TE and on a rocker to resuspend. NOTE: it generally takes two days for the DNA to resuspend at room temperature. After the DNA has been removed, tell the extractor to continue by scrolling to MAIN MENU 1, then press RESUME, NEXT STEP. The remaining steps in P4 prepare the machine for P5, which is its extensive purge. P5 takes over one hour to execute, and when this has finished remove and dispose of the run connectors, clean the top and bottom vessel clamps with dH2O and 100% EtOH, and shut off the extractor.

   Maintenance

   1. Shut purge: SHUT PURGE (formerly called TST5) is an extensive and powerful back-flush and purge (see Appendix IIB-4) that should be executed once a month or any time the extractor has been idle for more than 2 weeks. You must be present during much of the program. SHUT takes approximately 2 hours to run. Follow the SHUT procedure with an EPUR (Method 2 contains the programming for the SHUT purge [P6] followed by an EPUR [P5].) Follow the step directions in the manual.
   2. Vessel silanization: Vessels should be silanized every two to three months depending on its use. If the machine is used continuously the vessels will need to be silanized every 1-1/2 to 2 months (see section 6.2.13 of the manual). Use SurfaSil siliconizing agent to silanize the vessels (purchased from P.J.Cobert Assocoiates, catalog #42800, in 120 ml bottle). Run an EPUR prior to silanization, and follow with another EPUR or a SHUT PURGE if necessary.