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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/13


TITLE: 384-WELL E. coli LIBRARIES: CONDENSING FROM 96-WELL AND REPLICATION


Materials

Media-filled 384-well plates- Genetix (X7001) or Nunc 242757A [LS/18.1]
Sonicating water bath (Decon FS1006)
Decon 90
70% alcohol
Hybaid colony picking plate for gridder sterilisation CONS1001

Condensing a 96-well library to 384-wells using the PBA Gridder.

  1. Take the 96-well plates to be condensed out of the freezer to thaw.
  2. Sterilise the gridding tool. First ensure that it is loaded with the correct pins (96 thin (0.6mm) pins). If the pins need to be changed, place the tool in its plastic trough with the locking bolt pointing away from you, remove the existing pins and load it with the 96 thin pins. Carry the tool, in its plastic trough, to the sonicating water bath. The bath should be filled to the line with distilled water and a drop or so of detergent (Decon 90). Carefully place the tool in the clamp, and after making sure it is clamped securely, lower it into the sonicating bath, until it is submerged but the pins are not touching the bottom. Now the bath can be switched on (front bottom RHS). Set the time to ten minutes by first pressing the clock icon, then enter "10" and finally press the icon to the left of the numerals (a vertical line in a circle). The orange indicator light shows that the bath is now sonicating. DO NOT PLACE HANDS IN THE WATER OF A SONICATOR! After the 10 minutes are finished, switch the bath off, raise the tool and empty the water. This can be done by scooping the water out, or by using the siphon, and finishing by mopping up the water with paper towels. Now fill the bath with distilled water only, lower the tool back in, and sonicate for another 10 minutes.

At the end of this the bath is switched off, and the tool lifted out. Very carefully shake the tool to get rid of excess water. Now drench the tool using absolute alcohol in a wash bottle over the sink. Shake the tool again, and place in the drying cabinet for 30 minutes.

When the tool is dry, use paper towels to hold the handle which is now hot, and lay the tool on the bench to cool down before loading it on the robot.

Using the Flexys robot.

  1. Load the tool onto the tool holder. The end with the locking bolt goes towards the back, with the clip at the back pulled out to the left. When the tool is fully loaded, and is flush with the front of the holder, slide across the clip at the back to engage the bolt, so that the tool is locked in. Close the door.
  2. Double click the mouse on the gridder icon and the robot will switch on the correct main programme. The programme begins with the tool going to its home position at the back LHS and then back to the loading position. Make sure the laminar flow air supply box on top of the hood is switched on.
  3. Click on the file menu at the top of the screen and drag down till the "open" instruction is highlighted. When the saved files are displayed highlight the programme called 96cond.grd and then the programme that we want will be loaded. This programme uses 8 source plates (96-well) and 8 destination plates (384-well). The programme will make four copies of each 384-well plate, but this can be easily adjusted if required. The sterilisation steps are:
  4.   bath 3 (70% alcohol)    10 secs
      bath 4 (heater)          5 secs
      air dry                 15 secs


    NB. A quicker way to load the programme, if you have used it recently, is to highlight it when it is shown in the file menu, among the list of the four most recently used programmes.

  5. When you are satisfied that the programme looks correct try a dummy run by clicking on run programme. The programme should ask you if you want to start at spot 1, plate 1. If it does, click OK, if not (because a previous run was interrupted) reset the two figures to 1. Whilst the robot is doing its dummy run, check that it is doing everything, as expected, correctly.
  6. At the end of the test run, open the robot and fit the Hybaid plate for the alcohol bath. This is in the position in front of the heater. Make sure the plate is really securely in position before adding the 70% alcohol, which should 3/4 fill the bath. Spray the black plastic base on the LHS with 70% alcohol.
  7. Wear gloves from now on. Load the first 8 source plates into the rack. It is very important that the plates are clipped fully into the rack. When the rack is placed on the LHS position in the robot, check all plates are correctly loaded, by looking at them at eye level. An incorrectly placed plate will be higher than the correctly placed ones. Follow the plan below.
  8. Place the 384-well destination plates into the second rack. These 384-well plates are larger than the 96-well plates and are much stiffer fitting into the racks. It is essential that they are fully seated in the rack, otherwise they may move during the condensing of the library. Follow the plan below.
  9. Carefully remove the lids from the trays, placing them on the black plastic base on the LHS in the robot. Remove the lids starting with the plates furthest away, to avoid putting your arms over the open plates (and replace the lids, after gridding, in the opposite order). Remove the lid from the agar destination plate. Shut the door.
  10. Robot arrangement:

    Source Plates

    (96-well)

     

    Copy Plates

    (384-well)

     

     

     

     

    1

    5

     

    1.1

    2.1

     

     

     

     

    2

    6

     

    1.2

    2.2

     

     

     

     

    3

    7

     

    1.3

    2.3

     

     

     

     

    4

    8

     

    1.4

    2.4

     

     

    Heater

     

     

     

     

     

     

     

     

    EtOH

  11. Start the programme.
  12. Sometimes the programme will stop halfway through. Click the OK button, and the gridder will return to its start-up screen. Re-open the gridder programme. If the programme says 'speed out of range', press the abort icon and your original programme will be recovered. When you restart the programme you will be asked if you want to start at spot 'x'. Usually the robot will have already gridded from this spot, and if you have observed this it is safe to enter the next position and proceed from there. If you are not sure, then proceed from the suggested spot and the worst that will happen is that there will be a double transfer (of the same clones) in that position. Either way the tool will be sterilised before it goes to the next plate.
  13. Repeat this cycle, 7-9, for however many plates you are condensing and replicating.
  14. At the end of the session:

-refreeze the source plates
-incubate the 384-well plates. Wrap them in groups of eight tightly in cling film, and incubate at 37oC. Place a container of milli-Q water in the incubator as there is a problem with evaporation of media from 384-well plates.
-time permitting, re-sonicate the tool ready for the next person.

E. coli plates are incubated overnight at 37oC. Next day remove the plates, check that they have grown by carefully holding them to the light, sort out the different copy numbers, stack in groups of twelve, and place on polythene bags before freezing at -70oC. Archive, back-up, gridding and working copies of libraries should all be placed in different freezers, to guard against freezer failure.

Score one copy of the library for empty wells on the record sheets and file. Scoring is done by placing the grown plate on top of a blue or black food colour filled plate on a light box. Empty wells show as bright blue or black circles in the well. Growth in wells dulls the black or blue colour.


Replicating a 384-well library using the Flexys Gridder

  1. Fill the 384-well plates as above
  2. Thaw out the plates to be replicated
  3. Sonicate the tool head as described above
  4. Set up the PBA gridder as described above, but load the programme called 384cop.rep. This will make 2 copies of each 384-well plate. The programme can be modified to make a different number of copies if required.

Robot arrangement (for 2 copies per plate):

Source Plates

(384-well)

 

Copy Plates

(384-well)

 

 

 

 

1

 

 

1.1

3.1

 

 

 

 

2

 

 

1.2

3.2

 

 

 

 

3

 

 

2.1

4.1

 

 

 

 

4

 

 

2.2

4.2

 

 

Heater

 

 

 

 

 

 

 

 

EtOH

Proceed as for the previous description of using the gridder (steps 4 - 12) and incubate as before.


Replicating a 384-well library by hand

extra materials required:

384-well 'hedgehog'
Bunsen burner
methanol in large petri dish
disposable plastic tray
autoclaved milli-Q water

Fill the plates as above
Work in the E. coli hood. Spray clean with 70% alcohol before use
Wear gloves throughout
Place the burner on the RHS in the hood
Place the methanol dish on the LHS
To flame the 'hedgehog' dip in the methanol bath, REPLACE the lid on this methanol bath, and only then flame the 'hedgehog'. It is essential to keep the methanol covered and as far apart from the burner as possible.
Leave the 'hedgehog' on its side to cool down
Place the source plate and the correctly labelled media-filled plate in front of you, side by side, ensuring that both plates are orientated correctly with A1 at the top LHS corner. Lift off the lids.
Place the 'hedgehog' into the source plate, right to the bottom of the wells and move the pins from side to side.
Transfer the 'hedgehog' to the copy plate, and move the pins from side to side again to inoculate this plate.
[NB: when aligning the 384-well 'hedgehog' into a plate just look at the front row of pins, align them correctly, and the rest of the pins will also be in the right orientation.]
Place the 'hedgehog' into the plastic tray filled with milli-Q water to wash off the bulk of the cells.
Stamp the 'hedgehog' onto paper towels to partially dry it; then follow the methanol bath and flaming procedure described above.
Whilst the 'hedgehog' is cooling replace the lids on the plates you have just replicated and get the next set of plates ready.
If there is more than one copy to be made, then it is unnecessary to flame the 'hedgehog' between each of the copies of the same plate number - but only before you go into a new source plate.
Change the water bath frequently (tip the contents into a bucket of tegodyne)

Incubation is exactly as described above

The 384-well 'hedgehog' should be sonicated in the same way as the Flexys Gridder tool.


Guidelines prepared for CABRI by HGMP, December 1998
Page layout by CERDIC
Copyright CABRI, 1998

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