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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/1


TITLE: PRODUCTION OF HIGH-DENSITY GRIDDED YAC FILTERS


STANDARD OPERATING PROCEDURE

PRODUCTION OF HIGH-DENSITY GRIDDED YAC FILTERS

PURPOSE

To grow high density of YAC clones on nylon filters for in situ DNA preparation and hybridization screening.

DESIGN

  • GRIDDING OF YAC CLONES ON NYLON MEMBRANES
    PLACED ON GROWTH MEDIUM
  • INCUBATION FOR 26 HOURS
  • SPHEROPLASTING
  • LYSIS
  • NEUTRALISATION
  • TREATMENT WITH PROTEINASE K
  • WASHING OF FILTERS
  • DRYING AND BAKING

  • UV CROSSLINKING

 

WORKPLAN

Thur/Fri

1. Growth medium is poured in rectangular plates.

Fri/ Mo

2. Nylon membranes are labelled and placed on the surface of the medium.

Mo/Tu/We

3. Gridding of clones using Biomek 1000 or PBA robot..
5. Clones are gridded in a 4X4 array by transferring 1536 clones onto each filter from 16 microtitre plates.
6. Gridded filters are incubated for 26 hours at 300 C

Tu/We/Thur

7. Colonies are spheroplasted by treating filters with zymolyase.

We/Thur/Fri

8. Lysis is completed by treating the filters with NaOH-NaCl.
9. Filters are neutralised by submerging in Tris HCl-NaCl solution.
10. Filters are treated in Proteinase K solution.
11. After washing in Tris-HCl, the filters are air dried and baked at 800 C.
12. Finally the filters are UV-irradiated and stored.

BUFFERS,REAGENTS AND MEDIA


YPD agar + ampicillin (500ul of 50mg/ml filter sterilized stock /500ml YPD) + tetracycline (1.25ml of 5mg/ml stock /500ml YPD). Pour approx. 50ml per plate/filter
Ethanol
SCE buffer pH 7.4- 1M sorbitol, 20mM EDTA, 10mM Tris-HCL pH7.4, 14mM betamercaptoethanol (latter added last in fume hood)- 80-100ml/tray.
Zymolyase to be added to SCE before use. Final concentration 0.5mg/ml .
0.5M NaOH, 1.5M NaCl solution- 80-100ml/tray.
0.5M Tris pH 7.4, 1.5M NaCl solution- 700ml is enough for 120 filters.
50mM Tris pH 7.4, 0.15M NaCl solution- 1400ml is enough for 120 filters.
Proteinase K , (Sigma) - to be added to 600ml of the above solution before use. Final concentration
50mM Tris pH 7.4 - 700ml is enough for 120 filters.

MAJOR EQUIPMENT

Autoclaving facilities
Media preparation facilities
Sterile cabinet
300C Incubator
Biomek 1000 robot or PBA robot
Multi-pin inoculation tool
Sonicating water bath
370C Shaking incubator
Belly dancer
Hybaid oven
UV-crosslinker

OTHER REQUIREMENTS

Incubation trays with lids, (Dynatech) - Biobek only
Incubation trays with lids, (Hybaid) - PBA robot only
3MM filter paper, (Whatman)
Edding 1800 pen
Hybond N+, (Amersham , 0.45micron membranes, 7.8x11.9, 50/pack).
Perspex trays with lids
Plastic boxes
Pipettes and tips
Sterile glassware
Tweezers

DETAILED METHOD


POURING OF MEDIA

Add 500ul of 50mg/ml ampicillin (final concentration 50ug/ml) and 1.25ml of 5mg/ml tetracycline (final concentration 12.5ug/ml) to each 500ml of melted YPD agar (safety cabinet if possible)
Invert several times to mix.
Pour 50ml of medium per rectangular plate (ON A FLAT SURFACE, IN AS CLEAN AN ENVIRONMENT AS POSSIBLE).
Allow to set.
Cover with lids.
Store, right way up, at 40C.

LABELLING OF FILTERS AND POSITIONING ON AGAR PLATES (safety cabinet).

Use a Edding 1800 pen to label the top left hand corner of each filter.
The filter should be labelled as close to the edge as possible on one of the short sides.
The position of the label is used to orientate the filter and will correspond to position A1 of the colony grid.
If the filter is to be stamped with plates 1A-I it should be labelled '1A-2G+' . Other filters should be labelled using the same method.

The filters need to remain as clean as possible if they are to remain uncontaminated when incubated. Filters are therefore handled with tweezers sterilized by dipping in ethanol and flaming briefly. KEEP ETHANOL BOTTLE CLOSED AND AWAY FROM THE FLAME.

Using sterile tweezers place labelled filter onto prepared plate.
Ensure that there are no air bubbles between the agar and filter. Removal of bubbles can be acheived by gently lifting the edge of the filter with tweezers and repositioning the filter on the agar.

PREPARATION AND CLEANING OF GRIDDING TOOL- BIOMEK/PBA

Note: When not in use the tool should be stored in an ethanol reservoir to prevent damage to the pins. Examine the tool before use to ensure all the pins are straight. Bent pins will produce jumbled grids. Straighten or replace any bent pins.

The tool should be sonicated before use.
Fill sonicating waterbath with milliQ plus a splash of detergent. Fill up to the line at the top of bath.
Suspend tool over sonicating waterbath. Ensure the electrical parts don't get wet.
Switch on the bath. Set timer and sonicate for 10min. BE CAREFUL, NEVER PUT FINGERS IN BATH WHEN SWITCHED ON.
Switch off waterbath.
Empty bath with siphon pump.
Refill to same level with milliQ.
Sonicate for 2min.
Remove tool and squirt liberally with ethanol over sink.
Dry on its side in drying cabinet (approx. 10min or until completely dry).

GRIDDING OF COLONIES ONTO FILTERS (Biomek or PBA robot).

Thaw microtitre plates which are to be gridded. This will take approx. 1hour at room temperature.

Biomek procedure

Fit the gridding tool to Biomek.

Switch on computer, monitor, diskdrive, Biomek and flowhood.

At C:\ type: cd\ biotest3. Press RETURN.

Check tool transfer parameters (see separate note).

Type: grid. Press RETURN. The robot will home its motors, the screen will display prompts for the parameters, the number of replicas to make, the format of grid and the number of plates.

At the prompt for N, enter the number of replicas required from 1 to 6, (this will usually be 6). Press RETURN.

At the prompt G, enter the grid format 2x2, 3x3, or 4x4, (the YAC libraries are gridded in a 4x4 format). Press RETURN.

At the prompt S, enter the number of master plates to grid on each replica, (this is usually 16). Press RETURN.

Place a microtitre plate with wells 2/3 full of ethanol (approx. 200ul) in the sterilization position.

Place the agar plates with filters in the plate positions. Ensure that they are positioned correctly and do not slip around, if they do your grids will be jumbled. The label of the filter should be on the side facing towards you on the lefthand side. REMOVE THE LIDS.

Press RETURN, the robot will sterilize the tool.

Place the first microtitre plate in position (eg 1A). Position the plate so that well A1 is on the lefthand side closest to you. REMOVE THE LID.

Check that all lids have been removed and that all plates are positioned correctly.

Press RETURN. The robot will grid the colonies from this plate at position 1 on all the filters.

When the robot stops, remove the plate and replace it with the next (eg. 1B).

CHECK THAT THE LID IS REMOVED.

Press RETURN. The robot will grid the next set of cells at position 2 on the filters.

Repeat the above steps until the program is complete and the complete set of microtitre plates have been gridded onto the filters. ALWAYS CHECK THAT YOU HAVE POSITIONED THE CORRECT PLATE AND THAT THE LID HAS BEEN REMOVED.

Examine filters after gridding to ensure that each pin is gridding in the correct place and that innoculum is being transferred from each well.

In case of tool crash

If at a point where input is required from the keyboard, type: 90 and press RETURN.

If not, use EMERGENCY STOP on Biomek.

Switch of the robot and inspect the tool.

Straighten or replace any bent pins.

Turn on robot and start again.

You can resume form where you left off by entering A=x at the fit tool prompt, where x is the last plate you successfully gridded.

To change tool transfer parameters (if tool is not transferring from the bottom of wells).

Type CHECK. Press RETURN.

Place an empty microtitre plate in the master plate position.

Follow instructions and enter X,Y and Z values.

All pins should be in the centre of the wells and slightly lifted when the tool is in the plate.


PBA Procedure

INCUBATION OF PLATES

Plates to be incubated are stacked 3 deep, right way up, on a tray.
The tray is placed in a large plastic bag, sealed with tape, and placed in a 300C incubator for 26 hours.


PROCESSING OF FILTERS

1. Transfer filters, colony side up to a piece of 3MM paper soaked in SCE buffer + 14mM betamercaptoethanol + 500ug/ml zymolyase. Add zymolyase just prior to use and mix well. Each tray takes 12 filters. Air bubbles between the 3MM and filters should be removed by gently raising and lowering the filters.
Place lids on trays. Wrap in clingfilm.
Place trays in a large bag, seal and incubate overnight at 370C.

Note: This process should be carried out on the spheroplasting bench to minimise exposure to betamercaptoethanol.
Use the shallow perspex trays with lids which have been prepared for this purpose. Where filters are placed on buffer soaked 3MM, the paper should be moist all over, but excess solution should be poured off. If the paper is swimming in buffer the colonies on the filter will run into each other. Try and ensure no air bubbles are left between the tray and 3MM. A 5 or 10ml pipette can be used to roll out air bubbles and remove excess solution. Tweezers should be used when handling filters. The colonies are easily smudged.

2. Lay filters on 3MM soaked in 0.5M NaOH, 1.5M NaCl for 15min. Ensure there are no air bubbles. Colonies will become bleached as they soak up the solution. Lift and relay filters if non-treated patches become apparent during the 15 min.

3. Transfer filters to sheets of dry 3MM and air dry for 15min. . This step is important to obtain good filters.

4. Neutralize by Submerging filters in 0.5M Tris pH 7.4, 1.5M NaCl for at least 5min. DO NOT SHAKE.

5. Use tweezers to 'drag' the back of the filter over the side of the sandwich box to remove excess liquid and transfer filters one by one to 50mM Tris pH 7.4, 0.15M NaCl for 5min. DO NOT SHAKE.

6. Submerge filters in 50mM Tris pH 7.4, 0.15M NaCl plus 500ug/ml proteinase K for 30-60min at 370C with very gentle shaking.

7. Submerge filters in 50mM Tris pH 7.4, 0.15M NaCl for 5min with gentle shaking. Use belly dancer.

8. Submerge filters in 50mM Tris pH 7.4 for 5min with gentle shaking.

9. Rinse once more in 50 mM Tris-HCL pH7.4.

10. Use tweezers to 'drag' the back of the filter over the side of the sandwich box to remove excess liquid and any debris and air dry, DNA side up, on 3MM. When almost dry cover with a second sheet of 3MM to stop the filters curling up (NB. If the filters are too wet the colonies will stick to the 3MM and peel off the filters).

11. Bake in 800C Hybaid oven for 10 min. Filters should be placed in layers between 3MM.

12. UV irradiate , DNA side up on 3MM, in UV crosslinker. Switch on . Place filters inside.

Select OPTIMAL CROSSLINK. and START.

Store filters in plastic bags at 40C


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

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