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Materials

For agar plates

Bioassay plates [Nunc 166508 for PBA][Q-tray for Qbot]
LB agar
Kanamycin (25mg/ml stock)
Hybond N nylon membranes [Amersham NK9745 - special order 22.2 cm x 22.2cm ]
Forceps (blunt ended)
Sharp pencil to label filters
Gloves (Microtouch or powderless)

For gridding

PBA robot + 384 pin gridding head/orQbot + 384 pin gridding head
70% and 100% alcohol
Colony picking plates [Hybaid HB CPP]
Autoclaved 3MM paper cut to size
Gloves (Microtouch or powderless)
Cling film
37^oC incubator

For processing

Perspex trays
3MM paper cut to size of perspex tray
Sandwich boxes
Forceps (fine-pointed)
Timer
10% SDS
0.5M NaOH/1.5M NaCl
0.5M Tris pH 7.4/1.5M NaCl
50mM Tris pH 7.4/0.15M NaCl
2xSSC/0.1% SDS
2xSSC
50mM Tris pH 7.4
Shaking platform (x2)
UV Crosslinker
Plastic bags for storage (Ziplock)

Pouring the agar plates

PBA:

30 agar plates are poured on Fridays, for gridding runs on the following Monday and Tuesday.
30 agar plates are poured on Tuesday afternoon, for gridding runs on the following Wednesday and Thursday.
To avoid microwaving order the agar (8 - 10 litres) to be delivered hot on Fridays and Tuesdays. (Only 5 litres are actually required, but it is a good idea to build up stocks of agar which can be microwaved if there are problems with the Sanger autoclaves)

Qbot

24 plates are needed for each days gridding.
Order the agar as for PBA - but pour 24 or 48 plates at a time. In this case the person gridding has time during the run to pour the plates and lay on the filters for the next days run.

If the agar has been received from the Sanger kitchens before it has set, place the bottles directly in the hybridising oven at 65^oC. Otherwise microwave the 500ml bottles of LB agar. Make sure that the agar does not spill over in the microwave. The melted agar can be stored in a hybridising oven at 65^oC until ready to be poured.
Add 500µl of kanamycin for PAC filters
or
Add 600µl of kanamycin for cFugu filters to each bottle, once the agar has cooled down, just before pouring.
Two 500ml bottles will pour 7 Bioassay plates.
On PBA gridding runs 15 plates are gridded each day/Qbot runs use 24 plates per day
The PBA needs Nunc Bioassay plates/the Qbot needs Genetix Q-trays.

Only open the sterile Bioassay plates within the open fronted safety cabinet (sterile hood) in B310. The hood should have been sprayed with alcohol before use.
Pour 150ml of agar gently into each plate, using a sterile 50ml Falcon tube as a measure.
Use a sterile loop or sterile tip to push any air bubbles to the side of the dish before the agar has started to set.
Allow the agar to set, and also to dry after setting before replacing the lid. (If the lid is replaced too early a lot of condensation is created on the agar. This will result in filters that are too wet for gridding. Turn the plate upside-down as soon as the lid is placed on.)

Always pour 1 - 2 extra plates each time as spares to use if there are problems.

Store the plates, wrapped in clingfilm, upside-down, in the fridge until required.

Laying on the Hybond N paper

Work in a laminar flow cabinet, the 'hood', to lay the Hybond N filters on the agar.
Wear gloves.
Sterilise the flat ended, blue handled tweezers by dipping them in 100% alcohol and wiping dry. As the hood floor should have been sprayed with 70% alcohol and wiped, it is safe to lay the tweezers on this floor. Alternatively the filters can be placed on the agar by holding them in gloved hands provided that the gloves worn are powderless.
The membranes are usually labelled, placed on the agar and the plates wrapped in clingfilm in batches of three.
Lift the corner of the backing paper on the membrane, and label with pencil on the top left corner going downwards.

Use the sterile flamed blue-handled tweezers to lift each membrane and place on the surface of the agar. The membrane must be laid absolutely flat, with no air bubbles underneath.
The membrane is held above the agar, with the tweezers at diagonal corners, and laid down diagonally onto the agar.
It is essential that the membrane is square on the agar and central i.e. equidistant from each side.
This is something that gets easier with practice.
If the first attempt to lay the membrane down correctly is unsuccessful, lift the membrane up and start again (using the same membrane).
KEEP CALM!! (If thing get difficult - take a break, walk away, breathe deeply and start again!)
Examine the membrane from underneath - this is essential to ensure that there are no air bubbles between the membrane and the agar. (If there are air bubbles this means that the colonies will not be in contact with the agar, and without nutrient will not grow)
The plates are stored at 4^oC , upside down, wrapped in clingfilm, until required.
Membranes are usually put on three plates only at this stage. This allows the gridding to be started first thing in the morning. Once the gridding run has started the remaining filters can be laid on the agar.
Make a note of the batch number of the membranes used together with the date the plates are gridded and the number of filters gridded in the Filter Production Book.

Labels

PAC=7 filters per set               cFugu=4 large filters + 1 small
PAC 1 Plates   1 - 48               cFugu 1  Plates    1 - 48
PAC 2 Plates  49 - 96               cFugu 2  Plates   49 - 96 
PAC 3 Plates  97 - 144              cFugu 3  Plates  101 - 148
PAC 4 Plates 145 - 192              cFugu 4  Plates  149 - 196
PAC 5 Plates 193 - 240              cFugu 5  Plates  197 - 199 (only 3 plates)
PAC 6 Plates 241 - 288                         
PAC 7 Plates 288 - 321 (only 33 plates)   

(the PAC library has no plates labelled 174, 175, 176, 284, 285 and 311).
(the cFugu library has no plates labelled 97 -100)

These positions are replaced during gridding with empty sterile plates)

GRIDDING USING THE PBA ROBOT.

Remove the 384 well plates to be gridded from the freezer in B309 with the gridding copy of the library:

Lay the frozen plates on an alcohol cleaned bench to thaw completely before gridding.
Remove the 15 agar plates from the 4^oC fridge and allow to reach room temperature UPSIDE-DOWN before gridding. (as the plates warm up condensation will form. This must not stay on the membrane hence the requirement to be upside down)
Spray the 384 pin PBA tool with 70% alcohol and place in the drying cabinet
Turn on the PBA robot. Make sure that the computer, robot and controlled air supply are all switched on.
At the start-up screen select 1 - to start windows.
Form the Program Manager Screen:

The tool head will move
Use the mouse to hold on FILE and highlight:

Remove the 384 pin tool from the drying cabinet, and when cool load carefully onto the robot arm. The tool must be pushed right to the back and the retaining clip pushed across to hold the tool securely. The front of the tool will be flush with the robot arm when the tool is fitted correctly.
Make sure that the robot is fitted with the solid Bioassay plate holders

Place a Hybaid colony picking plate at the EtOH position and fill to just below the top with 96% Ethanol. The depth of the alcohol is important - IT MUST COVER THE PINS TO THE FULL DEPTH THAT THEY GO INTO THE TRAYS.
When plates are placed in the grooved holding positions it is essential that they are fitted tightly so check each one after placement. If the plate is loose then the gridding head will dislodge it further and the pins will not go in the wells correctly. This is the same for the alcohol bath - it must be fitted securely.
The alcohol must be replaced after each three filters have been gridded.
Wear gloves throughout.
70% Alcohol spray the black plastic area on the LHS in the gridder. This is where plate lids will go and must be clean.
Place the bioassay agar plates in the three positions taking great care that they are securely placed in the grooves. The labels MUST be at the top LHS.

Dry the tops of the 384 well source plates before stacking in groups of 5. So each time source plates are changed during the gridding run, two groups of 5 plates will be taken off, and the next consecutive two groups of five plates will be loaded.
Load the first 10 plates onto the gridder, taking care that plates 1-5 and plates 6-10 are in the correct positions. (see above diagram)
Remove the lids from the three bioassay plates, moving from right to left, and stacking the lids on the black plastic area on the LHS.
Remove the lids from the 384 well source plates, moving from right to left, and stacking the lids on the black plastic area on the LHS.
(moving from right to left minimises passing your non-sterile arms and hands over open plates - we want to avoid contaminating the plates once they have been opened.)
If there is any moisture on the top of the source plate wells at this stage it should be gently mopped up with sterile 3MM paper cut to size.

When everything is set up:

Gridding will start
At the end of 10+ minutes the gridding will stop and you will be prompted to load more plates

Carefully replace the lids on the 10 plates on the gridder (now moving from left to right)
Remove the 10 plates, and stack them on the bench in such a way that you are clear that these plates have been already gridded.
Load on the next 10 plates - taking great care that they are the next plates in the sequence, and that they are loaded in the correct positions.
Remove their lids, shut the robot door and click on OK in the prompt box to change plates

Gridding will resume
At the end of each 10 plates you will have to change the source plates, until you reach the last plates which will only be 8 in number - and which must be loaded into positions 1-8 on the robot.

When all 48 source plates have been gridded the first 3 filters have been prepared.
Replace the bioassay lids and remove the plates from the robot.
Wrap the plates in clingfilm and store at 4^oC.

Load the next three bioassay plates on the gridder and start the gridding run again.
In all there will be five complete gridding runs to do all 15 plates.

After gridding replace the source plates in their freezer trays and place on an empty shelf in one of the freezers. Next day return these frozen trays to their correct positions in the gridding freezer.

At the end of the day (as late as possible) place the plates in the 37^oC incubator. They should be incubated only in stacks of three, and should remain wrapped in clingfilm and upside down. Incubation should be for 12 hours, but overnight is longer than that - so remove from the incubator first thing in the morning.

Close down the PBA by exiting from the program and exiting windows. Switch off the Gridder, computer, screen controlled air supply and heater at the wall switches.

Sonicating the tool head.

Carefully take the 384 pin tool off the robot. 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 the shafts of the pins are submerged (but not the heads of the pins). The pins should not touch 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 robot to dry overnight.

Gridding cFugu 5 filters

There are three plates in the cFugu library left over when the gridding onto large filters has finished. These plates are gridded onto small Hybond N+ filters on Hybaid colony picking plates. cFugu 5 are gridded on the same day as cFugu 4, as they only take a short time to grid.
16 Hybaid colony picking plates have the same agar as the bioassay plates - but only 50ml/plate.
Small filters are used, labelled in the same way as the large filters, at the top LHS.

The solid bioassay base plates in the PBA are removed, and replaced be the gridding support base plates.

The PBA program to be selected is: PBA Flexys Gridder
The file to be selected is: fugusmal

Robot arrangement:

Load the 3 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.

Load the first 8 destination plates plate, with the Hybond membrane onto 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.

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.

Start the programme.
After the first 8 plates have been gridded remove these plates after replacing the lids and load the next 8 agar plates, remove the lids, shut the robot door, and run the gridding program again.

The 16 agar plates are incubated upside down, wrapped in clingfilm exactly as the large filters.

PLEASE RECORD THE DATE OF EACH GRIDDING ON THE RECORD SHEET PINNED TO THE SIDE OF THE PBA GRIDDER

PLEASE RECORD THE DATE OF EACH GRIDDING, FILTER SET, NUMBER OF FILTERS GRIDDED AND HYBOND MEMBRANE BATCH NUMBER IN THE FILTER RECORD BOOK.

Double spotting gridding design

All the large filters we produce are double spotted. This means that every plate is spotted twice onto the filter, in a set position. So a user will always get two spots come up for a positive hybridisation signal. The purpose of this is so that two spots makes very clear that the positive is really positive, and the gridding arrangement is such that each double spot has a distinctive orientation - making it very easy for the user to correctly identify the positive clone.

The spotting pattern is:

    3  5  6  8
    2  7  3  4
    6  4  8  5
    2  1  7  1

Please do not attempt to alter, or even check, the gridding arrangement in the PBA NEWPAC programme, as there is a fault in the software, which can lead to inadvertent erasure of the pattern.

GRIDDING USING THE QBOT ROBOT.

Gridding using the Genetix Qbot follows exactly the same principles as with the PBA Gridder - the main difference being that 24 filters can be gridded in a day leaving the operator time to pour out plates and lay filters for the following day.

Thaw out source plates
Whilst the plates are thawing spray 70% alcohol on the floor of the robot and wipe clean. Clean the linear drive runners.

Fill the alcohol bath with 96% Ethanol to just below the level of the bristles.

Loading the hotels

When the source plates are thawed gently wipe the tops of the lids. If the lids are wet this will interfere with the lid lifter.

Load the hotel:

Plates fit into the hotel with well A1 facing the open front of the hotel, and the labels showing.

Make sure that the plates are loaded in the correct order.

Layout of the Qbot "floor"

Q trays, with their filters on, are placed in the positions as shown above. They are held close together by the silver studs in the floor of the Qbot. Remove the lids before gridding starts.
Make sure that the filter label ** is in the correct position.
(When you stand at the LHS of the Qbot and load the Q trays you are loading them with the top of the filter furthest away from you)
Cleaning the Qbot, and setting up, takes about 15min.

Starting the Qbot

Switch on the compressor at the wall
The pressure should read 6 and 8 on the compressor and 80 on the pressure gauge on the robot when it is switched on.

Flick on the red and yellow button on the front LHS of the Qbot. This will activate the computer.
(Above this button, inside the Qbot, is a digital display which at this resting stage should read 0 0 2
At C:\> prompt type win

In Program Manager - open the window for 'Genetix Qbot'

For gridding select 'Genetix Gridder' and click OK

The message "Initialising Drives - Make sure main power is on" will be displayed

Switch on the green button  1    The indicator panel at the top of the robot will light up 
                            0    Power On
                                 Robot Working

Click OK

The "Make sure all doors are closed" prompt will appear (the robot will not work with the doors open)
The "Genetix sign on" screen will now appear:

Complete and click on 'gridding' box to proceed

Display now shows "Gridding Routine Set-up":

Type of spotting head       - 384 sprung
Spotting pattern            - Change to 4x4
Total number of filters     - 12
Fields per pattern          - 6
Number of stamps per spot   - 1
Enable Duplicator pattern   - X

Start-up Settings:

  First plate to spot    1
  First field to spot    1
  First pattern to spot  1
  First filter to spot   1

Sterilising Set-up:

  Number of cycles in bath  3
  Seconds in dryer          8
  Wait after drying         0

Additional options:

  Plate Stacker Enable      X     when using hotel

Gridding speed - fast

Gridding pin adjustment on filters -

When ready click on "Run"

Message will read

The run will now start - and will take approx. 2hrs 35min.

At the end of the first run, replace the lids on the Q trays and remove them. Fit the second batch of Q trays and remove their lids.
Change the 96% alcohol in the bath before starting this second run

At the end of the gridding runs

Click on "Close" and Quit from the "Genetix Sign On" screen

Switch off - by pressing the red button on the robot
1
0

Turn the big red and yellow switch to OFF

Switch compressor off at the wall

Remove the Sterilising Bath and empty out the alcohol
Soak the bath and brushes in 1/100 bleach dilution for at least an hour
Rinse with water and then soak in 70% alcohol for 15min.
Drain and allow to dry

Filters: Incubate and process exactly as for the PBA gridded filters

Processing the Filters.

Remove the 15/24 agar plates from the incubator
The filters are processed in batches - of 8 and 7 (for PBA) and three lots of 8 for Qbot gridding.. If the weather is warm it is best to put the unprocessed filters at 4^oC whilst the first batch are being processed (otherwise the filters will keep on growing).
Make up 1L of 0.5M NaOH/1.5M NaCl for 15 filters/ 2L for 24 filters
In all the processing steps, the filter is placed colony side uppermost.

All steps require gloves to be worn
Always handle the filters using fine forceps at the corners. Take great care not to wipe any of the colonies off the filters - hold at the very edge, using the margin where there are no colonies gridded.
Wear a lab coat with cuffs (or roll up your sleeves)

With practice, once the first batch of filters is at stage 4 (the first airdry) the next batch of filters can start to be processed. This allows 15 filters to be processed by 11.30am.

Prepare two large sandwich boxes and label as below:

From now on you will be transferring filters from box 1 to box 2 to box 1 etc. All the transfers will usually be in batches of 8
1 L of each buffer will be needed each time - an absolute minimum of 100ml/membrane is required.

Place the filters into a clean ziplock plastic bag. Write on the filter number, the date and the QA code of the filter .This code should also be entered in the filter record book.

Solutions Required

10% SDS

0.5M NaOH/1.5M NaCl

0.5M Tris pH 7.4/1.5M NaCl

50mM Tris pH 7.4/ 0.15M NaCl

This is a 1 in 10 dilution of the buffer above

20xSSC

2xSSC/0.1% SDS

2xSSC

1M Tris/HCl pH 7.4

50mM Tris/HCl pH 7.4

This is a 1 in 20 dilution of the buffer above [= 250ml of 1M stock + 4750ml MilliQ to make 5L final volume]

Filter Production - Record Keeping and Making up Sets

Records are kept of all filters made - so that if any user has a problem with a set of filters, we can look at the records and establish if there were any clues as to what may have gone wrong.

When handling the filters wear gloves

When the batch of filters have been cross-linked they are placed in ziplock bags, with the filter batch number and the filter number (i.e. Fugu 3) labelled on the outside of the bag. In the black record book details should have been entered in the first section called "Filter Production".

There are two parts to this:

1. Checking through newly produced filters after cross-linking to sort "good" filters from "not so good" filters.
2. Putting together "good" filters to make complete sets.

Good/NSG Filter sorting

Take the filters out of the bag and examine very closely to establish good (OK) and not so good (NSG) filters.

Divide the filters into good and NSG - and record this number in the record book

The filters are usually replaced in the ziplock bag, between two sheets of 3MM paper, with the good and NSG placed back to back, with 3MM in-between, clearly labelled with good and number of good filters written on the 3MM paper on one side, and NSG and number of NSG filters written on the 3MM paper on the underside of the bag.

Score all the filters like this.

To make up the sets:

Only use the good filters.

The number of complete sets that can be made will be determined by the filter which has the lowest number of usable good filters.
Lay out 3MM paper cut to size - for the number of sets to be made. Label the 3MM paper with the filter set number. This is determined by using the next consecutive number from the record book. PAC sets are preceded by the letter P, and Fugu sets by the letter F.
Lay the first filter (e.g. PAC1) on each pile - and record in the section in the record book "Filter Sets" the batch number of each filter used.
Lay the next filter (e.g. PAC2) on each pile - and again record the batch number of each filter.
Proceed until complete sets have been made.

Each set is placed in a ziplock bag and labelled with the Filter set number.

Before the sets are put away in storage, another member of the group must check each set to make sure that it is complete, containing the correct number of filters and that no filter is duplicated.

Several of the bags of individual filters will still have good filters remaining. Correct the number of good filters on the bag label. A separate record sheet is kept of this number of good filters remaining, but not yet in sets, and this helps us determine which filters to grid next.

Filters are stored at 4^oC in their ziplock bags

Dispatch - When filters are issued, please record in the "Dispatch record" section of the Filter Production book

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

© The CABRI Consortium 1999 - 2023
This work cannot be reproduced in whole or in part without the express written permission of the CABRI consortium.
Site maintained by Paolo Romano. Last revised on February 2023.