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LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/11


TITLE: 96-WELL YAC LIBRARIES: REPLICATION


Materials.

96-well plates: Falcon # 3072 (flat-bottomed)
Broth - YPD for CEPH MegaYAC library
SD for ICI and ICRF libraries
Ampicillin (see
GLI/1998/3/7)
Tetracycline (see
GLI1998/3/7)
500ml Duran bottles with 200ml glycerol. Sterile
Well-filler (Labsystems #831)
Sterile tubing cassette for well-filler
Sonicator
Decon
Absolute alcohol in wash-bottle
70% alcohol
Hybaid colony picking plate for gridder sterilisation
Hand replication also requires: SD or YPD agar + amp +tet
Petri dishes (Falcon #1058)
Hybond N filters (RPN119N)
'Hedgehog' (96 prong replicating tool)


Labelling the plates

Label the plates with coloured marker pens according to the following codes:

Blue for gridding
Black for working
Red for back-up
Green for archive (only very rarely required)


The plates are labelled on both the lids and the RHS of the bottom plate

Labelling on the lid should be as follows:

Code Date Plate

number

The codes that are used for each of the libraries are:

ICI - RA

ICRF - ICRF

CEPH - M


Filling the plates

Fri Use the well-filler in the Yac hood to fill the 90 labelled plates (2 copies of the 45 plates). Fill 120Ál/well with YPD + amp + tet.

Cover the plates well, with cling film or in plastic bags and store at 4oC over the weekend. Sterilise the tubing.

Week 2

Mon Use the gridder to make TWO copies of each of the 45 plates.

1. Load the tool with the 96 fat pins. Be sure that you put the pins in the correct position in the tool head (=top LHS of each set of 4 holes with the locking knob pointing away from you. Show Sarah or Kerstin if in doubt) Sonicate, first in weak detergent and then in milliQ. Shake, and drench in 100% alcohol and place in the drying cabinet. You need to do this first, so that the tool is dry before you load it on.TAKE CARE IT WILL BE HOT!

2. Take the CEPH Mega-Yac source plates out of the freezer. Use the back-up copy, take to the YAC hood and remove the plate sealers.

3. While the plates are thawing load the tool on the gridder, and select the programme I have pre-set called -- rep96x2.rep

4. Load up empty 96-well plates and the alcohol bath with 70% alcohol

Source Plates

 

Destination

Plates

 

 

 

 

1

 

 

1-G

3-G

 

 

 

2

 

 

1-W

3-W

 

 

 

3

 

 

2-G

4-G

 

 

 

4

 

 

2-W

4-W

 

Heater

 

 

 

 

 

 

 

EtOH

and start the programme to test that it is doing exactly what you want it to do

5. When you are satisfied that all is OK then load the first 4 source plates and the corresponding copy plates in the correct positions. Close the lid as soon as you have got the lids off. to try and maintain sterility.

6. Start replicating. Make sure that it is starting at - plate 1 copy 1

7. Load up the next trays, ready to swop over when the replication is finished.

8. When all the copying has been done, return the source plates to the back-up freezer, after replacing new plate sealers in the YAC hood.

9. Put all the copied plates on trays, cover in a large yellow bag including a tray of sterile milliQ and incubate at 30oC.

10. Unload the tool and sit it in its alcohol bath.

Wed Using the plate shaker, mix all the incubating plates and put back in the 30oC incubator.

Thurs Label your plates for next week - Blue for gridding

Black for working

Follow the list to see what colour to label which plates. Label 613 - 810 inclusive (= 80 plates)

Fri 1. Use the well-filler in the Yac hood to fill the 80 labelled plates.

Fill 120Ál/well with YPD + amp + tet.

Cover the plates well, with cling film or in plastic bags and store at 4oC over the weekend.

Leave the well-filler in the Yac hood, with the hood on. But rinse out the tubing with STERILE milliQ first.

2. Remove the 88 plates that you replicated on Monday (leave this till the afternoon if possible). Make up the required amount of bottles of 40% glycerol by adding YPD up to the 500ml level. THEN add the amp + tet to each bottle. Make up 3 x 500 ml bottles.

3. Check that the plates have grown. Plate shake each plate, and then add 120Ál/well of the 40% glycerol. Try to shake immediately before adding the glycerol so that the cells do not have time to settle.

4. Freeze the plates in their correct freezers, i.e. the gridding plates in the gridding copy - and ONLY THEN throw away the old plates you are replacing (blue bags for autoclaving.) AS YOU REPLACE WITH NEW PLATES CHECK THAT ALL THE SECTIONS HAVE THEIR FULL NUMBER OF 8 PLATES/SECTION.

Week 3

Mon Use the gridder to make ONE copy of the 80 plates that you filled on Friday.

1. Load the tool with the 96 fat pins. Be sure that you put the pins in the correct position in the tool head (=top LHS of each set of 4 holes with the locking knob pointing away from you) Sonicate, first in weak detergent and then in milliQ. Shake, and drench in 100% alcohol and place in the drying cabinet. You need to do this first, so that the tool is dry before you load it on.TAKE CARE IT WILL BE HOT!

2. Take the CEPH Mega-Yac source plates out of the freezer. Use the back-up copy, take to the YAC hood and remove the plate sealers.

3. While the plates are thawing load the tool on the gridder, and select the programme -- rep96x1.rep

4. Load up empty 96-well plates and the alcohol bath with 70% alcohol

Source Plates

 

Destination

Plates

 

 

 

 

1

5

 

1

5

 

 

 

2

6

 

2

6

 

 

 

3

7

 

3

7

 

 

 

4

8

 

4

8

 

Heater

 

 

 

 

 

 

 

EtOH

and start the programme to test that it is doing exactly what you want it to do

5. When you are satisfied that all is OK then load the first 8 source plates and the corresponding copy plates in the correct positions. Close the lid as soon as you have got the lids off. to try and maintain sterility.

6. Start replicating. Make sure that it is starting at - plate 1 copy 1

7. Load up the next trays, ready to swop over when the replication is finished.

8. When all the copying has been done, return the source plates to the back-up freezer, after replacing new plate sealers in the YAC hood..

9. Put all the copied plates on trays, cover in a large yellow bag including a tray of sterile milliQ and incubate at 30oC.

10. Unload the tool and sit it in its alcohol bath.

Wed Using the plate shaker, mix all the incubating plates and put back in the 30oC incubator.

Thurs Label your plates for next week - Blue for gridding

Black for working

Follow the list to see what colour to label which plates. Label 811 - 984 inclusive (= 82 plates)

Fri 1. Use the well-filler in the Yac hood to fill the 82 labelled plates.

Fill 120Ál/well with YPD + amp + tet.

Cover the plates well, with cling film or in plastic bags and store at 4oC over the weekend.

Leave the well-filler in the Yac hood, with the hood on. But rinse out the tubing with STERILE milliQ first.

2. Remove the 80 plates that you replicated on Monday (leave this till the afternoon if possible). Make up the required amount of bottles of 40% glycerol by adding YPD up to the 500ml level. THEN add the amp + tet to each bottle. Make up 3 x 500 ml bottles.

3. Check that the plates have grown well. Plate shake each plate, and then add 120Ál/well of the 40% glycerol. Try to shake immediately before adding the glycerol so that the cells do not have time to settle.

4. Freeze the plates in their correct freezers, i.e. the gridding plates in the gridding copy - and ONLY THEN throw away the old plates you are replacing (blue bags for autoclaving.) AS YOU REPLACE WITH NEW PLATES CHECK THAT ALL THE SECTIONS HAVE THEIR FULL NUMBER OF 8 PLATES/SECTION.

Week 4

Mon

Mon Use the gridder to make ONE copy of the 82 plates that you filled on Friday.

1. Load the tool with the 96 fat pins. Be sure that you put the pins in the correct position in the tool head (=top LHS of each set of 4 holes with the locking knob pointing away from you. Sonicate, first in weak detergent and then in milliQ. Shake, and drench in 100% alcohol and place in the drying cabinet. You need to do this first, so that the tool is dry before you load it on.TAKE CARE IT WILL BE HOT!

2. Take the CEPH Mega-Yac source plates out of the freezer. Use the back-up copy, take to the YAC hood and remove the plate sealers.

3. While the plates are thawing load the tool on the gridder, and select the programme -- rep96x1.rep

4. Load up empty 96-well plates and the alcohol bath with 70% alcohol

Source Plates

 

Destination

Plates

 

 

 

 

1

5

 

1

5

 

 

 

2

6

 

2

6

 

 

 

3

7

 

3

7

 

 

 

4

8

 

4

8

 

Heater

 

 

 

 

 

 

 

EtOH

and start the programme to test that it is doing exactly what you want it to do

5. When you are satisfied that all is OK then load the first 8 source plates and the corresponding copy plates in the correct positions. Close the lid as soon as you have got the lids off. to try and maintain sterility.

6. Start replicating. Make sure that it is starting at - plate 1 copy 1

7. Load up the next trays, ready to swop over when the replication is finished.

8. When all the copying has been done, return the source plates to the back-up freezer, after replacing new plate sealers in the YAC hood..

9. Put all the copied plates on trays, cover in a large yellow bag including a tray of sterile milliQ and incubate at 30oC.

10. Unload the tool and sit it in its alcohol bath.

Wed Using the plate shaker, mix all the incubating plates and put back in the 30oC incubator.

Fri 1. Remove the 82 plates that you replicated on Monday (leave this till the afternoon if possible). Make up the required amount of bottles of 40% glycerol by adding YPD up to the 500ml level. THEN add the amp + tet to each bottle. Make up 3 x 500 ml bottles.

3. Check that the plates have grown well . Plate shake each plate, and then add 120Ál/well of the 40% glycerol. Try to shake immediately before adding the glycerol so that the cells do not have time to settle.

4. Freeze the plates in their correct freezers, i.e. the gridding plates in the gridding copy - and ONLY THEN throw away the old plates you are replacing (blue bags for autoclaving.) AS YOU REPLACE WITH NEW PLATES CHECK THAT ALL THE SECTIONS HAVE THEIR FULL NUMBER OF 8 PLATES/SECTION.


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

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