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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/12


TITLE: PREPARING 4x4 GLYCEROL PLATES OF YAC CLONES


Materials Required

YAC plates from the gridding copy of the library
Sonicator
Decon 90
Absolute alcohol in washbottle
SD agar in Hybaid colony picking plates (for ICI library)
YPD agar in Hybaid colony picking plates (for CEPH MegaYAC library)
Hybond N pre-cut membranes
Blue handled tweezers
Absolute alcohol bottle
Bunsen burner
Glass 'hockey stick'
3MM paper, pre-cut, sterilised
50% glycerol in SD or YPD broth + ampicillin + tetracycline
Hybaid colony picking plates


Method

Preparation

Preparation

At the moment, the only libraries that are being gridded in this way are the ICI and CEPH MegaYAC libraries.

The agar plates can be poured in advance and stored in the 4oC cold cabinet. (see GLI/1998/3/2 for method)

  1. Thaw out the required plates from the gridding copy of the library. 16 source plates are gridded onto one 4x4 gridded filter, so you will be thawing out the source plates in groups of 16.
  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, placing the pin corresponding to well A1 in the top left hand hole of the tool. (This is a temporary measure until we get the three separate tools). First 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.
  3. 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.

  4. Working in the YAC laminar flow cabinet, the 'hood', label the required number of Hybond filters. Wear gloves. Sterilise the flat ended, blue handled tweezers by dipping them in 100% alcohol and flaming them. REMEMBER TO KEEP THE ALCOHOL AND NAKED FLAME AT OPPOSITE CORNERS OF THE HOOD. As the hood floor should have been sprayed with 70% alcohol and wiped, it is safe to lay the tweezers on this floor.

Take out a membrane and lay it on its backing paper. Label, using the Edding 1800 pen. on the top LHS of the membrane, as below:

X
X
X

When all the Hybond membrane have been labelled, use the sterile flamed blue-handled tweezers to lift each membrane and place on the surface of the agar in the Hybond colony picking plate. The membrane must be laid absolutely flat, with no air bubbles underneath. The Hybond N membranes are susceptible to having lots of very small air bubbles underneath. To remove these, put the glass hockey stick into a beaker of absolute alcohol, and flame to sterilise. When cool the hockey stick can be run repeatedly over the membrane squeezing all the air bubbles to the edge and out. It is easier to do all the membranes at the end, and there will be no need to resterilise the hockey stick. Do not use the tweezers to remove air bubbles, as these scratch the surface of the membrane and thus the very small colonies on a 4x4 grid will not grow well.


Using the PBA 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 4x41glyc.grd and then the programme that we want will be loaded. This programme uses 16 source plates and one destination plate, in a 4x4 grid pattern with the 96 fine pin head.
    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. 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.
  8. Place the destination plate, with the Hybond membrane into the first position on the second rack. These colony picking plates are larger than the 96-well plates and are much stiffer fitting into the racks. But it is essential that they are fully seated in the rack, otherwise they may move during gridding and the gridded filter pattern will be very haphazard.
  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

     

    Gridded Plates

     

     

     

     

    1

    5

     

    X

     

     

     

     

     

    2

    6

     

     

     

     

     

     

     

    3

    7

     

     

     

     

     

     

     

    4

    8

     

     

     

     

     

    Heater

     

     

     

     

     

     

     

     

    EtOH

  11. Start the programme.
    After the first 8 plates have been completed, the programme will ask you to load the next source plates (9-16). Remove the plates 1-8 after replacing the lids, load plates 9-16, remove the lids, shut the robot door, and press the OK icon. The robot will now complete gridding the second half of the gridded filter.
  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 spot (of the same clone) on 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 gridded membranes you are preparing.
  14. At the end of the session:
  15. -refreeze the source plates
    -incubate the gridded membrane plates
    -time permitting, re-sonicate the tool ready for the next person.


    Incubating the gridded membranes.

    Place the agar plates, upside down, wrapped in cling film, in the 30oC incubator for two days.


    Placing the membranes on paper soaked in glycerol.

    1. Add an equal volume of SD or YPD broth to a Duran bottle of autoclaved glycerol. Add ampicillin and tetracycline to the required final concentration. E.g.;
      100ml glycerol
      add 100ml broth
      + 200Ál ampicillin
      + 500Ál tetracycline
    2. Work in the YAC 'hood'. Pour some of the 50% glycerol into a sterile large petri dish. Sterilise the blue-handled forceps by flaming in alcohol, and then use them to remove a sheet of pre-cut, autoclaved 3MM paper and soak the paper in the glycerol. Lift out the paper with the forceps and allow the glycerol to drain off. Just before it has stopped dripping lay the paper into a Hybaid colony picking plate, taking care to ensure that there are no air bubbles underneath the paper. If there are air bubbles, the sheet can be lifted off and relaid.
      The glycerol amount can be adjusted if it is incorrect, by either draining off excess glycerol by tipping the plate, or by adding extra glycerol if the paper is too dry.
    3. Label this plate, on the lid, with:
    4. the library name
      the date
      plate numbers contained on the grid

      Also place a red line around the lid and the base in the LH corner.

    5. Hold the agar plate with the gridded membrane, and carefully lift the membrane off using the forceps. This is best achieved by using forceps to lift up the RHS of the membrane, lifting the membrane off the agar and dragging it across the plate until you can grasp the LHS of the membrane with the second pair of forceps. Carefully place the membrane onto the glycerol soaked paper, laying it in such a way as to prevent air bubbles underneath. When using the forceps to lift the membrane you must be very careful not to touch any of the colonies. If there are any air bubbles trapped, try lifting the membrane and relaying it.
    6. The plates are now ready to be frozen, RIGHT SIDE UP, in the -20oC freezer. Throw away the old 4x4 plates that you are replacing.
    7. The final job is to complete new record sheets and to place them in the appropriate folder.


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

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