PrimerStation 960 Operating Instructions

PrimerStation 960

Check gauges on left side of machine to make sure all lines are appropriately pressurized.
Check dew point. Start drying chamber if dew point is above -25^oC: Tools - Macro - Dry System 2 hr. (You can cancel dry macro at any time by pressing Abort at the main screen).
Check liquid N₂ level - at least 1/8 dewer or don't start.
Check acetonitrile (ACN) wash level in cycletainer - need at least 9 liters for run of 25 mers.
Check ancillary reagent levels:
- TCA/deblock (red tubing) - need ~2 in for 25 mer run.
- Oxidizer (orange tubing) - need ~1.25 in for 25 mer run.
- Cap A (yellow tubing) - ~1.25 in for 25 mer run.
- Cap B (blue tubing) - ~1.25 in for 25 mer run.
  *If changing bottles, decanting ancilliary reagents into new bottles is OK. They are all less sensitive to humidity than phosphoramidites.
Mark reagent levels with tape. Date new bottles.
Check or replace:
- Tetrazole/activator - need ~1/2 bottle for 25 mer run. Remember bottle seal. Screw bottle snugly into machine.
  *If pooling bottles, use molecular sieve, and date bottle. Maximum capacity of 5 g sieve: pooling 2 full 450 ml bottles.
- A, C, G, T phosphoramidites - 40 ml ACN for 2 g of phosphoramidite powder, or 100 ml ACN for 5 g. Use 18-20 gauge needle with 60 ml syringe (16 gauge too big). Blow dry N₂ into syringe 3-4 times to get rid of moisture. After last purge, expel N₂ to 40 or 50 ml mark. Insert needle into ACN bottle, invert, and equalize pressure with bottle until 40 ml is drawn into syringe. If bottle is slightly pressurized when needle is withdrawn, be careful of squirting ACN. Snap phosphoramidite bottles into machine. Do not pool when changing; always use fresh bottles and remember to date. Phosphoramidites are very sensitive to humidity.
  *If ambient humidity is relatively low (e.g., during winter), it is OK to uncap and decant ACN directly into phosphoramidite bottle. In general, dissolved phosphoramidites last for ~4 weeks. Ancillary reagents should be changed if machine is left idle for 4 weeks.
From main screen, press Protocol button. Select Rocker-3.pro.
Select Tools - Macro - Prime Monomers - peer into chamber with flashlight and check to see that four troughs on right are filling, ie., that fluidic lines are cleared for pumping and draining.
*This step is not essential if phosphoramidites were not changed for this run.(~2 min)
Select Tools - Macro - Prime Ancillaries - peer into chamber with flashlight and check to see that common trough on left does not overflow.
*This step is not essential if ancillary reagents were not changed for this run.(~1.25 min)
From main screen, press Load Sequences button. Select your group (.gpf) file. The machine will automatically proceed to load tip trays. Load the 4 trays in succession, ignoring tray detection error (just press return). After all tips are loaded, do NOT verify each pin.
Check that each of the 96 plungers has a tip: Tools - Maintenance - Carriage - press Home. Peer into chamber with flashlight. Sight down all 12 rows and check to see that all plungers have been loaded with tips.
*If tip did not load onto plunger, note position (e.g., B11), and determine correct tip by loading your .grp file in Group File Viewer. After you determine the correct tip, load that tip on an empty tray, and load that specific plunger: Tools - Maintenance - Door - press Open Door, put in tray, close door, select Carriage tab, move carriage to either the 4, 8, or 12 position to align piston with correct plunger, select Pins tab, actuate correct plunger, and eject empty tray. Repeat: remove empty tray. An empty tray will block the tips from touching the reagents during a run.
Mark all reagent levels. You will want to know how much of each of the 9 reagents is consumed for this run. Or, if reagent levels do not change after a run, you know that pumping was not successful and to discard that set.
READY TO RUN: check dew point. If too wet (> -25^oC), Tools - Macro - Dry. Enter into log book:
1. starting dew point
2. name of group file
3. starting N₂ level
4. starting ACN wash level
Press START from main screen. Machine is now on automatic (~11 hrs for 25 cycle run).
Refill the 4 trays with the appropriate tips.
*If CPG (controlled porosity glass) dust can be seen in the plunger hole of the tips, blow in N₂ to get rid of any settled CPG.
*If insufficient tips to fill the trays, you can run Group File Viewer, load your sequence .gpf file, see the first base for each position, and manually assemble a tray of tips.
After run: measure reagent usage to 0.1 in accuracy. Write in log book.
Get 96 well polypropylene plate (see below). Write name of oligo set on plate. Using multichannel pipettor, fill each well with ~160 ul of NH₃OH.
Unload Tips from main screen. Insert empty tray.
*If you need to manually unload tips, insert empty tray. Then Maintenance - Carriage tab - Eject position. Then select the Press tab, and click the Press button twice (down/up) to eject tips off plungers. Remove tray by going to Door tab.
Write down the positions of any lost tips!
Place tips into NH₃OH. Agitate tips to get rid of any bubbles, overcome surface tension. Cleave DNA oligos off CPG for 1 hr, 23^oC (room temp).
Make sure heat block is on at 60^oC. Make sure you have Costar 96 well plate storage mat seal for the next step.
Agitate tips with fingers again. Remove tips from NH₃OH and place face up in hood for residual NH₃OH to evaporate before discarding. Mark empty spots (ie., lost tips) on 96 well plate seal.
With multichannel, add ~60 ul of NH₃OH into each well to make up for what evaporated during the 1 hr cleavage. Cover plate with Costar plate sealer. Put into blue aluminum fixture with rubber gasket top. Lock down with screwdriver, 1/4 turn. Deprotect for minimum 5 hr at 60^oC on heat block (usually can leave overnight).
Cool down plate enough to handle (5-10 min, 4^oC). Unscrew off blue aluminum top. Be careful of cross contamination by NH₃OH condensed on plate sealer when pealing off top.
Place blue aluminum air dryer on top of plate. Use small amount of air flow (10-15^o CCW on knob). Dry for 45 min, 60^oC. Cover plate with new Costar plate sealer (old one will be deformed from NH₃OH fumes).

DONE!

Regular Maintenance Procedures

Changing ACN (wash) Cycletainer

Turn off both valves (Liq and Gas) on tank.
Undo quick-disconnect fittings: push in button and pull bottom of fitting up.
Disconnect digital level cube meter.
On new tank: attach green tube to gas; blue tube to liq. Connect level cube.
Open valves on both ports.

Changing N₂ Dewer

Turn off outlet valve on dewer and shutoff valve on regulator.
Screw off pressure regulator with wrench (regulator stays with lab). You may need 2 wrenches to prevent unscrewing of outlet on dewer.
Attach regulator to new dewer, turn on dewer valve, and regulator valve. Adjust pressure regulator, if necessary, so that output is 100-110 psi. If pressure gauge on dewer gaspack (ie., vapor pressure inside dewer) is below 100-110 psi, turn on the PB (pressure builder) valve on the gas pack.

Emptying Waste Drum (Also see IAS printed instructions) - Right now, the machine does not differentiate between halogenated and non-halogenated waste.

Drum is about 56 liters, so get 2-3 yellow translucent Nalgene 20 liter liquid disposal containers.
Turn off vacuum pump: Tools - Maintenance - Gas Valves. Off is red.
Make sure ball valve (green) on waste tank is in off position (horizontal). Isolate waste drum by shorting vacuum line: drums are connected in serial on the vacuum line so just detach the line from one in-union connector and reattach to the other in-union connector.
On ACN cycletainer, turn off green knob connected to green N₂ (7 psi) line. Disconnect the quickfit connector and then unscrew (green) line from fitting. Route hissing, pressurized line to waste drum.
Remove 1/8" waste-in line (yellow for non-halogenated) to drum, and connect the green N₂ line to pressure tank.
Connect a waste tube to the green ball valve and route other end into Nalgene container. To better contain volatile waste fumes, wrap Parafilm around opening of Nalgene container and puncture with waste tube. Turn on ball valve and drain (~11 min to fill 20 liter container).
Fill out disposal tag, use preprinted labels. Fill out date, building, room, investigator, phone no.
Bring filled Nalgene container by door.

Changing the 6 Valve Filters (left side of machine) - Whenever waste tanks need to be emptied, also change or clean filters.

Get two 5/16" wrenches and pair of tweezers.
Depressurize chamber by opening door.
Change filters one at a time to avoid mismatching fluid lines. Unscrew the filter body from the line using wrench if too tight. Leave alone the downstream fittings to the valves.
Remove filter with tweezer and discard. If you do not have new filters, rinse with ACN squirt bottle. Also rinse unscrewed filter body with ACN. There should be no grittiness from CPG when screwing back together. (Cut filters to length if too long, using old filter for correct length).
Filter fits into the large end of the body with closed end showing. Finger tighten filter body back together (should be pretty tight or it will leak).
Screw filter body back into waste line. Orientation of filter is:
chamber - large end of body - small end - valve (imagine as arrow pointing in direction of fluid flow).

Calibrating Dew Point Sensor - Every few months.

Unscrew purple spot sensor probe from metal block with wrench. You can leave the hissing metal block attached to the exhaust line. Expose sensor to room/ambient humidity and let equilibrate for a few mins.
Using mini flathead screwdriver, adjust wet knob on dew point meter to 0^o. Unplug meter cable from sensor. Adjust dry knob to -100^o.
Reassemble metal block.

Miscellaneous Procedures

Opening Chamber

Dry chamber for 10 min (purge with N₂) to minimize vapors.
Quit Primerstation program (tray door opens).
Unscrew plate off door.
Undo the 8 hex screws holding front panel (1 turn).
To replace front panel: Line up dogs so that rounded side will latch with a clockwise (from outside) turn. Turn 90^o with long arm of allen wrench inserted into hex screw. Feel for latching. Then with short arm of wrench, tighten screws with another 90^o turn.

Taking Out Carriage

Undo the 2 thumbscrews attaching carriage to drive belt (behind carriage).
Undo the 8 screws that hold the two rail brackets to chamber walls.
Take out carriage + rails.
Make sure homing pin clears troughs when positioning carriage + rails in chamber. Rest carriage on troughs.
Position front two brackets first, then the back two. Tighten screws.

Cleaning Troughs of Precipitates

Using ACN squirt bottle: open door and squirt onto troughs to dissolve crystals.
Tools - Maintainence - Fluid Valves tab. Drain A, C, G, T, moat, common non-halogenated valves.

Machine Storage - (If machine will not be used for a while)

Replace tetrazole, A, C, G, T bottles with ACN bottles. Tetrazole crystals and phosphoramidites can gunk up the fluid lines if machine is idle for long periods. Run ACN through lines: Maintenance - Fluid Valves tab - Mon (A,C,G,T) buttons or Activator button.
Leave ancillary reagents, ACN wash cycletainer, N₂ dewer attached to machine.
Shutdown Windows 95 and turn off machine.
When starting up again: run the tomer.gpf sequence file with ACN in tetrazole, A, C, G, T lines.

Group File Viewer - preparing sequence files for PS960

Run Group File Viewer from Start menu.
Tools menu - Open Custom File. Select your ASCII text file containing your 96 sequences. Look at any one of these files as an example for the format to use. Basically, the gene name is commented out and the next line is the sequence 5' to 3'.
After the program converts your file and color codes the bases, go to File - Save Group File. Save the converted, PS960 readable .gpf file. Remember to include the .gpf extension. The directory to save it in on our machine is Desktop\Shortcut to Seq Folder. *Note: .gpf files are also just text files, so you can view them with Word Pad or Note Pad. "Primer Station Group File" has to be found on second line of file.
Press Exit on the main screen.

Troubleshooting

Symptom: High dew point in chamber. Possible causes:

Impeded gas flow

Brass fitting of gas exit from chamber corroded
HDPE tubing to building exhaust kinked

Synthesis reagents contaminated with water

How to test if ACN wash is not anhydrous enough: dry chamber to around -40^oC, fill common trough with ACN, let sit for ~1 hr, and see if there is an increase in dew point. If it is above -25^oC, ACN is probably contaminated or there is breach in ACN line.

Machine has been sitting open/idle for a long period of time

Teflon and other materials inside the machine absorb water. So when you start the machine up after a long period of being idle, it could take several days to a week to completely rid the chamber of water. For dry continuously for 48 hrs, go to Tools - Macro - Dry 48 hr. If machine has been open/idle during humid weather (eg., summer months), it could take even longer to dry.

Reagents and Supplies

Polypropylene 96 Well Plates

Plate

29444-104

Storage Mat Cover

29445-122

Liquid N₂ Dewer

MedTech Gases

GP240

Contacts

Acetonitrile (ACN) Wash Cycletainer

Mallinckrodt Baker (higher quality ACN)

Part# JT9019

Doe & Ingalls
25 Commercial St.
Medford, MA 02155

American Bioanalytical: 800-443-0600 (Jean, sales) or Tom Hellman, Sales Rep, tom@americanbio.com, (617) 513-0299, cell phone. Call for pickup of old and delivery of new. Ready to ship 56 liter acetonitrile cycletainers (catalog no. AB00120-56000): $559/tank and $25 shipping (charged for one way/ship in only). Must bring old tank down to basement dock for pickup; new tanks come straight to WAB 342A. 200 liter tanks also available for future reference.

Phosphoramidites and Ancillary Reagents

Glen Research: 800-327-GLEN (Fax: 800-934-2490)

Cap A, acetic anhydride in ACN, 2 l: 40-4213-62, $115.20
Cap B, ACN/methylimidazole/pyridine, 2 l: 40-4223-62, $115.20
Oxidizer, 0.05 M iodine/water/pyridine, 2 l: 40-4130-62, $130
Deblock, 3% TCA/dichloromethane, 2 l: 40-4140-62, $57.60
Activator, 1H-tetrazole in anhydrous ACN, 450 ml: 30-3110-57, $60
Diluent, ACN, 100 ml: 40-4050-50 $6.40
dA-CE phosphoramidite, 5 g: 10-1000-50, $75
dC-CE phosphoramidite, 5 g: 10-1010-50, $75
dG-CE phosphoramidite, 5 g: 10-1020-50, $75
dT-CE phosphoramidite, 5 g: 10-1030-50, $75

Contacts

Robert Steen - director of the HHMI Biopolymers Facility where our machine is, rsteen@genetics.med.harvard.edu
Dave Boccuti - product manager of the PS960 at Intelligent Automation(IA), boccuti@automationonline.com
Rick Neves - engineering/technical contact for the PS960, Rick@GenomicTechnologies.com
Josh Ormsby - mechanical designer/field engineer at IA, josh@automationonline.com

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