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  2. Synthesizer common question and troubleshooting

Here is a list of frequent questions relating to the MerMade instruments which you may find useful.  If you need further assistance do not hesitate to contact us via a service ticket.

Machine will not power on

  • Open the panel or control box for the machine. If the power supply is on or items such as the fan are on this may be an indicator that you have blown a fuse.  Check the black fuse box and replace with similar type fuse if necessary.  Clean up spills or exposed wires that may have caused a short in the system and try again.
  • (M192) Be sure that the E-stop has not been activated.

Software is not communicating with the machine

  • Ensure the machine is on and that all cables are connected from the computer to the instrument. (M4, M6, M12) The blue National Instruments cables can at times become loose at the machine. Move the cable around so that the pins adequately make connection.

Pressure leak / Argon supply not lasting long

  • Be sure that the Argon Supply being delivered to the machine is below 25 PSI. Most of our equipment is fitted with a 25 PSI pressure relief valve that will vent gas out if the pressure is above this threshold.  This is for safety purposes and BioAutomation generally recommends the pressure be in the 10-20 PSI range.
  • The most common location of a pressure leak is a loose amidite or reagent bottle cap. Double check to make sure all bottles are secured tightly and that the caps are not visibly cracked and that the O-ring is not compromised in any way.  Replace any O-rings as needed.

Liquid not dispensing

  • Check to make sure that the liquid you are dispensing is in the correct bottle position and that that position is receiving adequate pressure.
  • Our machines are set to cut power to the valves when the liquid sensor has been activated. Be sure that the liquid sensor is dry (green light) before attempting to fire any valve.
  • At the bottle, make sure the liquid line is inside the liquid and that any particulates or Trap Paks are not interfering with the flow rate.
  • Check to see if the valve for that bottle is firing by either using the Injection Head test screen or the Test I/O screen in the software. You should usually be able to hear a sound, whenever the valve is fired.  Repeat this step several times, as valves that are not used often may need several attempts before the solenoid starts firing again.  Tapping the valve lightly several times may also be useful for valves that have not been used in a while. If still no sound is heard, replace the valve and see if that solved the issue. (Refer below on how to replace a valve)
  • If the valve fires but still no liquid is being delivered check the liquid line from the valve to the injection head and the line going from the bottle to the valve. You may see some crystallization in the lines or the line may be kinked preventing flow outwards.  Replace the line and test again.

How do I replace a valve?

  • Liquid Valves
    1. Depressurize any bottle that may be feeding liquid to that valve or place an empty bottle in its position.
    2. Fire the valve so that any excess liquid is removed from the lines or manifolds.
    3. Detach the 2-pin connector from the valve or remove the wire from the terminal blocks using a small flat head screw driver if applicable.
    4. Remove the screws from the old valve and place the new valve in its position.
    5. If manifold mounting the valves, be sure that any O-rings are in place on the manifold
    6. Tighten just enough for a good seal and no leakage, as overtightening may lead to deformation of the solenoid. (If a torque screw driver is available the recommend force is 4-6 in-oz.)
    7. Reconnect any pin connector or wires.
    8. Return pressure or liquid to the bottle position and confirm function.
  • Vacuum Valve
    1. Once you have located the troublesome valve drain any liquid in the line and/or manifold if possible.
    2. Disconnect the 2-pin connector going to the valve.
    3. For drain valves that are not manifold mount, disconnect the liquid lines going to the valve.
    4. Unscrew the old vacuum valve and place the new valve in its position
    5. Reconnect the 2-pin connector and drain lines and confirm function.

Liquid line dispensing slowly/leaking

  • If the liquid is dripping slowly from the line when you attempt to dispense it, there may be particulates or kinks in the liquid line leaving the valve that are preventing it from flowing outwards. Replace the line if possible and test again. 
  • If the liquid is still dispensing slowly, or the valve drips constantly, even when you are not firing it, you will need to replace the valve.
  • In warmer temperatures, 90F (33C), it is common for the Deblock reagent valve to drip because of the out gassing of the DCM.

Inconsistent liquid dispensing

  • Calibrate the machine to ensure that the liquid volume you want to dispense, is being dispensed, per protocol.
  • If the bottle pressure has recently been changed the machine will require recalibration.
  • Ensure that the pressure supply going to the bottle position has sufficient flow either by adjusting the needle valve or removing any kinks in the line.
  • The liquid valve may be failing and will require replacement. (This is more common for the acidic reagents such as deblock)
  • If the valve still dispenses different volumes you may have a gas supply leak or a faulty regulator. (Refer to pressure leak section above)

Clogs in liquid/waste lines

  • This happens often when amidites positions are not used for a while. BioAutomation recommends that if a line is not intended to be used for a while to wash that position with Acetonitrile. Replace lines if crystallization cannot be removed.
  • Some amidites or modifiers, such as O-Methyl-G, are more likely to crystallize than others. This can usually be reduced by the use of a co-solvent.

Column position not draining / draining slowly/ inconsistent (M4, M6, M12)

Note: Not all column positions or columns are going to drain at the same rate.  Be sure to only use these troubleshooting techniques if positions vary drastically or if oligo quality is being affected.

  • Ensure the column is pressed in all the way. Columns that are not properly sealed will not get sufficient flow through them.
  • Replace the column. Not all columns are packed the same and therefore some columns may be more inhibiting to the flow than others.
  • Fire the vacuum valve for that position using the Test I/O screen. If you do not hear the valve fire you will likely need to replace it.
  • Inspect the column chuck to make sure there is no blockage in the chuck. Remove blockage or change column chuck if that is the case
  • Replace the drain line leaving the column chuck. If you find this difficult to do or have questions please contact BioAutomation. 
  • Replace the vacuum valve if it varies drastically from all the other column positions and the above troubleshooting techniques did not work.
  • Inspect the flow cell and drain valve. Either one could have an obstruction causing a slow drain rate.
  • The vent line leaving the vacuum pump may not have adequate flow outwards. This will cause the flow of vacuum to vary throughout the run steps leading to inconsistent draining. Ensure the vent line is not kinked or inhibited.

Column plate not draining/ draining slowly (192, 192E, 192X)

  • If you are not using a full plate be sure that any unused positions are sealed well with a plate sealer as to prevent any vacuum leaks.
  • If only a few column positions do not drain the CPG in these columns may be too tightly packed. Replace the columns and try again.
  • Check to make sure that the gasket is in place and that the toe clamps are compressing the plate on top of the gasket
  • Make sure the waste carboy is sealed tightly and that the fittings are not loose.
  • Ensure that there are no kinks in the vacuum or waste lines. The vent line leaving the vacuum pump must also be free of kinks to help increase the vacuum flow rate.

Columns/plate drains even when vacuum is not being applied.

  • If the plate or column position drains quickly as if the vacuum valve is open, the valve may have failed and could be stuck open. This will usually require replacement.  (Refer to valve replacement section above)
  • If the column or plate drains on its own very slowly there may be a small leak somewhere preventing an adequate seal. Check to make sure all columns gaskets and plates are sealed correctly. 
  • If the column or plate drains slowly and there are no leaks there may be residual vacuum left in the line from the previous run step. Increasing the equalize time may solve your issue.

Trityl monitors not functioning correctly (M4,M6,M8, M12)

  • Smaller scales such as 50 nm and oligos with lengths greater than 50 bases will be more difficult to detect because of lower yields. Be sure this is not the case before contacting BioAutomation.  Refer below if you are having yield issues. 
  • If you are experiencing a lot of false-positives (Trityl is detected but full length product is not being made) the LED on your optical sensor has likely gone out and you will need a new sensor. Contact BioAutomation if this is the case.

Slide Not Moving

  • When powered, the slide will not move if the lid is open. Ensure that it is closed all the way before moving the slide in the software.
  • Any spills that may have occurred could be inhibiting the slide movement. Clean up any spills and lubricate the slide or bearing rail.
  • The software may have lost communication with the slide. Ensure that the cables going from the machine to the computer are connected.

Slide making a loud noise

  • Turn off power to the machine and lubricate the slide and/or bearing rails using Krytox. Restore power to the machine and see if any difference in the noise level has been made.
  • Check to see if the slide has passed one of the limit sensors and is hitting the hard stop. If adjusting the limit sensor has failed to solve the problem, contact BioAutomation to purchase a replacement.
  • If none of the above steps work, the slide or motor may need to be replaced. Contact BioAutomation for a solution.

Software Freezing

  • Your log folders may be too big and that is making it difficult for the software to write a log file to that folder. Archive any old log files.
  • The computer may be running low on RAM or hard drive space. Remove any unnecessary files or software. Adding more RAM may also be beneficial.

Poor oligo quality/yields

  • Ensure fresh reagents are on the machine. Once on the machine, Amidites generally have an expected lifetime of 1-2 weeks while reagents have an expected lifetime of 1-2 months.  Call us for more information about Amidite and Reagent life on the machine and in storage.
  • Calibrate the machine to make sure that the correct volumes are being delivered for the script file. It is important to inspect both the liquid calibrations and the vacuum calibrations for the specific script file you intend on using.  If the bottle pressure or vacuum has recently be changed this will require a recalibration.
  • Make sure that the chamber is sealed to prevent moisture from seeping into the chamber and affecting the oligo quality. Our machines have been tested in environments up to 25% humidity and it is not recommended that the humidity go above this percentage.
  • Poor yields may be an indicator of bad cleavage. Ensure that the Ammonium Hydroxide or AMA is relatively fresh before use and that adequate cleavage times are allotted based on the chemical you decide to use.

How do I calibrate liquid delivery for the instrument?

ALL INSTRUMENTS

  1. Ensure the reagents and amidites are on the machine and bottle positions are fully pressurized. Acetonitrile may be substituted for the amidite positions as this will help conserve amidite and the viscosity does not very greatly from amidites in solution.
  2. Prime the lines so that they are filled with liquid
  3. Go to the calibrate valve screen
  4. Open the injection head or door and place a collection tube underneath the line for the valve you wish to use.
  5. In the software, select the valve you wish to fire and the volume you wish to dispense. If the volume you want to dispense is not there add it along with an estimated time for how long the valve should be open.
  6. Hit inject to collect the liquid in the tube.
  7. Use a pipette or scale to measure the amount of liquid dispensed.
  8. Change the valve open time until it delivers the amount of liquid you need.
  9. Repeat for all valves you intend on using and hit save.

ONLY FOR MM192, 192E, 192X

  1. For instruments that have group injections you will need to calibrate the group injections as well by going to the calibrate G injections screen.
  2. Choose the group you wish to calibrate.
  3. Place a strip of 8 or 12 pcr tubes underneath the lines and hit inject to collect the liquid.
  4. Adjust the individual valve times until they all deliver the correct amount of liquid.
  5. Repeat for all groups and hit save.

How do I calibrate vacuum pulses for the instrument?

ONLY FOR M4, M6, M12

  1. Go to the calibrate vacuum screen.
  2. Place a fresh unused column in one of the positions you intend to use.
  3. Hit inject for liquid delivery and pay attention to the liquid as it drains through the column.
  4. After all the pulses and prior to the final drain a little bit of liquid should still remain about the CPG in the column. Adjust the vacuum settings until this is the case. (Do not set vacuum valve open time to be <20ms as the valves are not rated for open times below this
  5. Ensure that the final drain evacuates all remaining liquid from the column and adjust as needed.

ONLY FOR 192E, 192X

  1. Place a filled column plate in the machine.
  2. Go to the calibrate vacuum pulse screen.
  3. Select the correct amount of columns in the plate and the correct amount of liquid to dispense (You can usually substitute acetonitrile for all reagent calibrations as this will not render the columns unusable afterwards)
  4. Adjust the vacuum pulses until a sufficient amount is pulsed through the columns without liquid being completely evacuated.
  5. Ensure that the final drain evacuates all the liquid from the columns.