Robot LI-COR Cycle Sequencing

Robot LI-COR Cycle Sequencing

Protocol for Web Scientific Membrane Combs on LI-COR

Loading volumes for Epicentre labelled samples: 64-well comb ~0.6l per tooth,

96-well comb ~0.2 l per tooth.

  1. Clean plates and insert in electrophoresis chamber.
  2. Put 400ml 1 x TBE in bottom tank.
  3. Attach top buffer tank.
  4. Put 540ml deionised water in top tank then add small amount 10 x TBE, from the 60ml needed to give 1 x TBE, and mix well.
  5. Flush well area.
  6. Attach electrodes.
  7. Close chamber door.
  8. Wait for interlock chimes.
  9. Select relevant run conditions * in electrophoresis settings.

*For 66cm (0.2mm), using Version 2 collection software;

Pre-run; Volts: 10.0, Current: 1.0, Power: 1.0, Time: 00:10,

Run; Volts: 3000.0, Current: 30.0, Power: 75.0, Time: 12:30,

Temp: 45.00C, Scan speed: 3-Moderate.

  1. Start run and wait till gel temperature reaches 45OC. (This time can be used to load samples on to the comb).
  2. Check run status is “Waiting to load samples”.
  3. Open chamber door.
  4. Remove upper electrode.
  5. Flush well area.
  6. Overlay with 300l 10% Ficol/Blue Dextran.
  7. Replace electrode.
  8. Close chamber door.
  9. Wait for interlock chimes.
  10. Start run. Allow 45 sec from the time the high voltage light shows on LI-COR.
  11. Press “Pause run”, and wait until command executed.
  12. Open chamber door.
  13. Remove upper electrode.
  14. Insert comb till touching gel surface.
  15. Replace electrode.
  1. Close chamber door. - Run should automatically restart.
  2. Allow 25-90 sec* after voltage registers in status window, [*shorter times give tighter, less tear-drop shaped bands]. If wait < 25 sec may encounter problems pausing run to remove comb.
  3. Press “Pause run”. Wait until command executed.
  4. Open chamber door.
  5. Remove upper electrode and comb.
  6. Flush well area.
  7. Add the rest of the 60ml of 10 x TBE to the top tank and mix well.
  8. Replace electrode.
  9. Close chamber door. - Run should automatically restart.

Robot LI-COR Cycle Sequencing

Handling Fluorescently labelled substances:

IRD labels primers and reaction products are sensitive to visible light. To avoid prolonged exposure to light:

Store primer stock tubes in the dark.

Keep primer stock tubes in black boxes for use on benchtop.

Load final reaction products on to sequencing gel as soon as possible after reaction completed, or store at –200C immediately.

Protocol:

Thaw the reagents on ice. N.B. It is critical to vortex mix all components to ensure uniform composition of the solutions before dispensing.

  1. Start cooling relevant robot positions.
  1. Prepare working dilutions of primers from stock solutions.
  1. Prepare Master Mix as follows - volumes are in l and are as quoted in Epicentre protocol for manual processing of 1 sample. (See also * in Step 7 below).

N.B. When calculating the volume of Master Mix required for robot set up remember to allow at least 12l per reaction, (14l if primer included), + 1l per needle, (for waste volume), + 15l (as residual volume).

Buffer 7.2

Water 1.8

Enzyme 1.0

10.0

  1. Load plasticware and place reagents in relevant positions on robot platform.
  1. Start script and add 5.5l template DNA when prompted.
  1. Complete the robot script, including the cycler protocol, (see later).

N.B.2l primer solution *, and 2l termination mix added during robot script. * If only one primer pair required, the relevant amount can be added to the Master Mix – remember to delete the relevant steps from the robot script and increase the Master Mix aliquoting volume to 14l.

  1. Remove template DNA plate and completed reaction plate from the cooled stacker. Manually add 3l stop/loading buffer.
  1. Denature/reduce samples prior to loading on the sequencing gel, (see later).

Robot Thermal Cycler Protocols:

These protocols were developed for MWG Primus thermal cyclers. If a non-MWG cycler is used it may be necessary to modify cycle conditions. Ramping time is very important. Non-optimum ramping may lead to poor quality data.

Sequencing Reaction Protocol:

  1. Within the robot script, after the 96-well plate is placed in the thermal cycler, the cycle sequencing reaction is performed using the following protocol, (saved as LICSEQ.CYC in robot PCR editor):

Close cycler lid with 200N.

Heat lid to 1100C.

Hold 950C for 5 minutes.

Start Cycle Loop (30 cycles)

Hold 950C for 45 seconds.

Hold 500C for 20 seconds.

Hold 700C for 1 minute 15 seconds.

End Cycle Loop.

Hold 80C for 2 minutes.

Deactivate lid heating.

Store for 5 minutes at 80C.

Open cycler lid.

  1. If necessary, the reactions are stored in the cooled stacker at 80C, or a fridge at 40C, overnight.

Denature/Reduce Protocol:

  1. After manual addition of 3l stop/loading buffer the 96-well plate is placed on slot P1 and the Denature_Reduce.rss script performed, (includes the following protocols, saved in robot PCR editor as Denatred.cyc and Red.cyc respectively):

Close cycler lid.

Heat lid to 950C.

Hold 950C for 5 minutes.

Deactivate lid heating.

Hold 80C for 45 minutes at 80C.

Open cycler lid.

  1. Within the robot script the compression pad is removed and sample volume reduction performed as follows:

Close cycler lid.

Hold 950C for 5 minutes.

Open cycler lid.

  1. Within the robot script the 96-well plate is moved to the cooled stacker, then manually removed to freezer at -200C.
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