SPHEROPLASTING YAC POOLS

LABORATORY PROCEDURES FOR GENOMIC LIBRARIES

REFERENCE NO: GLI/1998/3/3

TITLE: SPHEROPLASTING YAC POOLS

Materials Required

Autoclaved 500ml, 250ml and 100ml Duran bottles
Autoclaved 1.5ml Sarstedt tubes or 15ml conical Falcon tubes
Yeast pools
Test Sarstedt tube with 50µl line marked on
Vacuum system with autoclaved glass pasteur pipettes
Sterile bijou bottles or universal tubes
Eppendorf 0.5ml combitips and dispenser
Plastic weighing boats, large size
Plastic spatula
70% alcohol
50ml Falcon tubes
37^oC shaking incubator
46^oC water bath
5mM EDTA, pH8 [see separate SOP for solutions]
Zymolyase, 100T [ICN #320931]
b -mercaptoethanol
Low Melting Point Agarose [Gibco 5517UB]
Spheroplasting Solution [see SOP GLS/07, and also below]
YLS [see SOP GLS/07]
Microwave oven (Toshiba, ER-8730)
Green sieve lids for 50ml Falcon tubes

Solutions

Spheroplasting Solution	x40 = 40 secondary pools @ µl/blob = 4 x 10 primary pools @ 50µl/blob	x20 = 20 secondary pools @ 40µl/blob = 4 x 5 primary pools @ 50µl/blob

Spheroplasting Solution	500ml	250ml
Zymolyase 100T	50mg	25mg
b -mercaptoethanol	500µl	250µl

2% Agarose

LMP Agarose	1g	0.5g
in Spheroplasting solution	50ml	25ml
b -mercaptoethanol	50µl	25µl

NB: When making a different number of pools into 'blobs' the volumes of the two solutions will have to be adjusted.

Method

Wear gloves throughout.
All tips, bottles, pipettes etc. must be sterile .

Preparing the cell pellets

Primary Pools:	200µl cell pellet required, in 15ml Falcon conical tubes
Secondary Pools:	50µl cell pellet required, in 1.5ml Sarstedt tubes

Remove the required cell pools from the 4^oC cold cabinet. Transfer 1.6ml of the thoroughly resuspended cells into the labelled 1.5ml Sarstedt tubes using a P100 pipette, or 9.6 - 11ml into the labelled 15ml Falcon tube. Spin the Sarstedt tubes at top speed, 13,000rpm, in the MSE microcentaur centrifuge, for 1 min.

Check the volume of the pellet by holding it up to the test Sarstedt tube which has a line drawn round it corresponding to 50µl. Use the vacuum system, with a sterilised glass pipette, to aspirate the supernatant carefully from each tube. It is important not to touch the actual cells. Place the pasteur pipette in a spare clean 50ml Falcon tube when not in use.

The volume of the pellet observed will determine how much more of the yeast suspension needs to be added to each tube, to achieve an approx., 50µl size cell pellet. This additional volume is usually in the range of 0.4 - 0.8ml for the Sarstedt tube.

With the 15ml tube, used for the primary pools, it is easier to see the 200µl mark, as this is marked on the side of the tube. These tubes have to be spun in the Sorvall T6000B bench top centrifuge for 4 min at 2700rpm (setting 5), with the appropriate bucket adapters. If the first spin does not produce the required 200µl cell pellet, more cell suspension will have to be added, as with the 50µl sample above.

Once the required cell pellet volume has been achieved, the tubes are stored in the 4^oC fridge for use on the following day. The supernatant is left on top of the cells overnight.

Preparing the solutions

Turn on the water bath, at 46^oC, after having checked that it has sufficient water inside.
Make up the required volume of spheroplasting solution and 2% agarose, as shown above.

Spheroplasting solution

Choose the volume required. If only 250ml is needed, pour out that volume into a sterile 250ml Duran bottle to the correct level. For 500ml, adjust the volume to the 500ml mark, using a sterile pipette, if required. Carefully weigh out the Zymolyase and add to the solution. The Zymolyase does not immediately dissolve, so the bottle must be shaken gently, until it does. Add the b -mercaptoethanol to the solution, in the fume cupboard. Label the bottle clearly, so as not to confuse it with the stock bottles that do not have the additional Zymolyase and b -mercaptoethanol.

2% agarose

Use a sterile pipette to add the required volume of spheroplasting solution from a stock bottle, not the one that has just been prepared to a sterile 100ml Duran bottle. Mark the level of liquid on the outside of the bottle using a marker pen. Weigh out the LMP agarose, add to the bottle, and mix. Loosen the lid and place the agarose in the microwave, setting 3 for 1 min. This will have to be repeated until the agarose has dissolved. Take care that the mixture does not boil over. When dissolved, hold the bottle using the red rubber 'hot hand', and take to the fume cupboard to add the b -mercaptoethanol. Back in the laboratory, check the mark on the side of the bottle, and if necessary add more spheroplasting solution, using a sterile pipette to bring the volume up to the correct level. Place the agarose in the 46^oC water bath, with a red lead weight on top of the lid, to prevent the bottle floating.

Spheroplasting the cells

Spin the tubes containing the cell suspensions, as described above. Aspirate the supernatants, again taking care not to touch the cell pellets. Add 1ml of the spheroplasting solution (+zymolyase and b -mercaptoethanol) to each 1.5ml tube; or 4ml to each 15ml tube for the primary pools.

For each sample :

Place a sterile bijou bottle (for secondary pools) or a sterile universal bottle (for primary pools) into the 46^oC water bath, taking care that the bottle does not float away. Using a sterile tip, or sterile 5ml pipette, add 1ml (or 4ml) of the agarose, as appropriate, to the bottle. Without changing this tip/pipette, mix up the cell suspension and add to the agarose, mixing well.
Place a weighing boat (250ml size) onto ice in the ice bucket. Label the corner of the weighing boat with the correct label for the pool number that you are currently using.
Place a 0.5ml combitip in the dispenser, setting 5 for primary pools and setting 4 for secondary pools. Fill the combitip with the cell agarose mixture, and dispense 'blobs' onto the cooled weighing boat. Make sure the 'blobs' are not placed so close together that they mix. The combitip will have to be filled several times before the cell agarose mixture has all been dispensed. Each primary pool will need 2 weighing boats.
Transfer the weighing boat from the ice to the bench, ready for the next sample.

Repeat steps 2-5 for each cell pool, remembering to use fresh tips/pipettes/combitips each time .

Allow the 'blobs' to cool - 30min 4^oC or 1hr RT.
To each weighing boat, use a sterile pipette to add 10ml of spheroplasting solution (+zymolyase and b -mercaptoethanol). For the primary pools you will need to add a total of 40ml (i.e.; 20ml per weighing boat).
Use the plastic spatula, sterilised in 70% alcohol and wiped dry between each pool, to gently scrape the 'blobs' into a sterile labelled 50ml Falcon tube. Each secondary pool will be put into a 50ml Falcon with 10ml of spheroplasting solution, and each primary pool will be in a 50ml Falcon with 40ml of spheroplasting solution.
Place all the tubes into polystyrene racks. Secure the racks in the 37^oC shaking incubator, taped so that they are held at a 45^o angle. Incubate at 37^oC, shaking (setting 140rpm) for 2 hours.

Lysing the cells

Place two green sieve lids in a beaker of autoclaved MilliQ water.
For each 50ml Falcon tube, remove the top, screw on the green sieve lid, and drain out the spheroplasting solution into a large beaker. Vigorously tap the tube on the bench to dislodge any of the blobs that may have remained stuck to the green sieve lid. Place the green lid into the water to be rinsed. Alternate the use of the two lids. Place the Falcon tube lid lightly onto the tube, and replace the tube in the polystyrene rack.
When all the tubes have been drained of the spheroplasting solution, add 10ml of lysis buffer, YLS, to each tube (40ml for the primary pools) using a sterile pipette. There is no need to change the pipette between tubes. The pipette can be used to help push any 'blobs' that are stuck to the side of the tube down into the buffer. Tighten all the lids.

NB. if the YLS has precipitated due to a low external temperature, it can be redissolved by warming to 37^oC .

Incubate, as before, at 37^oC, tilted, for 30-60min.
Drain the tubes, as step 1-3, replacing with fresh YLS and incubate at 37^oC, tilted, overnight.
Next morning, repeat step 5, replacing with fresh YLS and incubate at 37^oC, tilted, for 2 hours.

Storing the agarose pellets ('blobs')

Drain off the final YLS wash, using the green sieve lids as before.
Using a sterile pipette add 5mM EDTA to clean, sterile labelled universal tubes (for the secondary pool 'blobs') or 50ml Falcon tubes (for the primary pool 'blobs'). Add 10ml to the universal tubes, and 40ml to the Falcon tubes.
Use the plastic spatula, sterilised in 70% alcohol and wiped dry between each pool, to gently scrape the 'blobs' into these filled tubes.
Store the 'blobs' in their correct labelled racks in the appropriate 4^oC cold cabinet.

Guidelines prepared for CABRI by HGMP, December 1998
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