TITLE: PRODUCTION OF HIGH-DENSITY GRIDDED YAC FILTERS
STANDARD OPERATING PROCEDURE
PRODUCTION OF HIGH-DENSITY GRIDDED YAC FILTERS
To grow high density of YAC clones on nylon filters for in situ DNA preparation and hybridization screening.
BUFFERS,REAGENTS AND MEDIA
Incubation trays with lids, (Dynatech) - Biobek only
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)
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 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.
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.
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.
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.
INCUBATION OF PLATES
Plates to be incubated are stacked 3 deep, right way up, on a tray.
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.
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
© The CABRI Consortium 1999 -