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Yeast microarray using the Genisphere 3DNA Array 900 kit

By Andrew Armstrong,2014-06-20 18:48
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Yeast microarray using the Genisphere 3DNA Array 900 kit

    Yeast microarray using the Genisphere 3DNA Array 900 kit

    David B. Kushner and Benjamin J. Tiede ’05

    Department of Biology, Dickinson College, Carlisle PA 17013

    It needs to be noted that the GCAT webpage already has several useful protocols for both working with yeast, using Genisphere kits, and handling microarrays. Under “General Resources” see Mary Lee Ledbetter’s 2003 ISB Workshop report (“3DNA method”) and Todd Eckdahl’s 2003 tips on working with microarrays (“Technical Tips”). Under “Yeast Resources” see Karen Bernd’s “Making Media and Growing Yeast,” and “Isolating Yeast RNA/mRNA” as well as Todd Eckdahl’s “3DNA Method for Making Probes, Pre-Hybe, Hybe, Wash for

    Microarray Hybridization.” These are excellent resources and these were referred to heavily

    upon developing this protocol. Note that the protocol below uses the acid phenol method of yeast RNA isolation (Bernd’s uses the spheroplast method) and uses lifter cover slips for the microarray hybridizations.

    DBK wishes to also note that he received lots of invaluable help and advice on methods from Laura Hoopes and Anne Rosenwald.

    The key to successful microarray work is being prepared. Several things MUST be done ahead of time, both in terms of acquiring materials and reagents, and for each “step,” having water baths, incubators, etc. turned on.

    NOTE that when ordering the Genisphere 3DNA kit, ask for reverse transcriptase to be provided. Genisphere’s RT is fine for this work, but is separate from the labeling kit. However, you will need to buy DTT (see section G). Alternatively, you can buy Superscript II RT from Invitrogen (which comes with DTT), but it is expensive.

Outline of contents of this Protocol:

    A. Yeast growth media

    B. Plasmids

    C. Preparing competent yeast cells (for transformation)

    D. Transformation of competent yeast cells

    E. Growing yeast in liquid media for RNA isolation

    F. Isolation of RNA from yeast using heated, acid buffered phenol

    G. Preparation of cDNA from total yeast RNA using components from the

    Genisphere 900 kit

    H. cDNA hybridization

    I. Post cDNA hybridization wash/Cy dye hybridization

    J. Post Cy dye hybridization wash

A. Yeast growth media

    As noted above, in the Yeast Resources page of the GCAT website, Karen Bernd has supplied information about growing yeast. Those of you not familiar with yeast will quickly learn that

    there are many subtly different ways that yeast can be handled. We report here the ways we grow yeast.

    If you simply want to grow yeast under different conditions (in other words, you do not want/need to transform yeast with plasmids) or simply need to grow yeast to prepare for prepping transformation-competent yeast cells, “complete media” is used.

    1. Complete media (YPDA). This is a simple mixture of yeast extract (Y), peptone (P),

    D-(+)-glucose), and adenine (A). The adenine helps prevent the dextrose (D; also known as

    yeast from “pinking,” and is optional. However, I always use adenine.

1a. Complete liquid (YPDA) media, 500ml:

In one 1L bottle, add 250ml ddHO, 5g yeast extract, and 10g peptone. 2

    In a second 1L bottle, add 250ml ddHO and 10g dextrose. 2

    Autoclave, cool to about 50?C, then, working near flame (sterile technique), combine and add 4ml adenine (1g/200ml ddHO, filter sterilized). Mix well. 2

1b. Complete solid (YPDA) media, 500ml:

This is good for about 20 plates (100x15mm Petri dishes):

In one 1L bottle, add 250ml ddHO, 5g yeast extract, and 10g peptone. 2

    In a second 1L bottle, add 250ml ddHO, 10g dextrose, and 10g agar. The agar will not go into 2

    solution until autoclaved.

    Autoclave, cool to about 50?C, then combine and add 4ml adenine (1g/200ml ddHO, filter 2

    sterilized). Mix well. Pour plates working near flame (sterile technique). After solidified, turn plates upside down and store at room temperature (RT) overnight. Bag plates agar-side up; plates can be stored for several months at RT.

    2. Minimal media (synthetic defined (SD)). Such media is needed if yeast are to be transformed with plasmids (for plasmid selection/retention). Working with minimal media can be initially expensive (in order to buy all the amino acids needed to make “dropout powder”). However, prepared minimal media can be purchased (for example, CLONTECH/BD Biosciences sells this, and I imagine other sources are available) and may be worthwhile if, for example, working with yeast is to be a once-yearly lab exercise.

    Dropout powder: When making a dropout powder, if you plan on transforming in one plasmid with a leucine marker, add all the amino acids EXCEPT leucine to the dropout powder. If transforming two plasmids, one with a lysine and one with a uracil marker, add all the amino acids EXCEPT lysine and uracil to the dropout powder.

Amino Acid Grams

    Adeninesulphate 4

    Arginine 2

    Aspartic acid 10

    2

Glutamic acid 10

    Histidine 2

    Isoleucine 3

    eucine 6 L

    Lysine 3

    Methionine 2

    henylalanine 5 P

    Serine 40

    hreonine 20 T

    Tryptophan 4

    Tyrosine 3

    Valine 15

    Uracil 2

    Add one amino acid at a time to a mortar and grind to a fine powder with a pestle. Add to a 250ml bottle.

    Upon adding each mashed amino acid to the bottle, shake bottle well to obtain an even distribution of amino acids.

2a. Minimal liquid media, 500ml

    O, 3.5g yeast nitrogen bases WITHOUT amino acids, and To a 500ml bottle, add 100ml ddH2

    0.7g of the appropriate dropout powder.

    In a 1L bottle, add 400ml ddHO and 10g dextrose. 2

    Autoclave, cool to about 50?C, then, working near flame (sterile technique), combine into the 1L bottle. Mix by gentle swirling.

2b. Minimal solid media, 500ml

To a 500ml bottle, add 100ml ddHO, 3.5g yeast nitrogen bases WITHOUT amino acids, and 2

    0.7g of the appropriate dropout powder.

    In a 1L bottle, add 400ml ddHO, 10g dextrose, and 10g agar. The agar will not go into solution 2

    until autoclaved.

    Autoclave, cool to about 50?C, then combine into the 1L bottle. Mix by gentle swirling. Pour plates working near flame (sterile technique). After solidified, turn plates upside down and store at RT overnight. Bag plates agar-side up; plates can be stored for several months at RT.

3. Galactose media.

    Sometimes yeast need to be grown in galactose, not dextrose. For example, if you transformed in a plasmid that allows you to inducibly drive expression of a gene via a GAL1 promoter. In

    such cases, simply omit dextrose from the above formulations. Then, after autoclaving and allowing media to cool, add 20% galactose (filter sterilized do NOT autoclave galactose) to a

    final concentration of 2%. For example, when making 500ml media, omit 50ml water from one of the bottles so that after autoclaving and combining, add 50ml 20% galactose to make a final volume of 500ml with galactose at 2%.

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B. Plasmids

If needed, plasmids for transformation into yeast do not have to be “ultrapure” – in other words,

    no CsCl banding needed. From 50ml o/n 2xTY bacterial culture, a traditional P1 P2 P3 solution plasmid prep with RNase step is sufficient. Minipreps are generally at too low a concentration for convenient use in yeast transformations if transforming 2 or more plasmids at once.

C. Preparing competent yeast cells (for transformation)

There are MANY ways this can be done. Zymo is but one company that sells a “quick-

    transformation” kit that works well for one or two plasmids, but the efficiency is not as great as making cells competent via the protocol below, which is easy and can be done in a few hours.

    1. Using sterile technique (e.g. working near a flame), use a toothpick to scoop up a pinhead amount of yeast from a solid plate and drop toothpick into a 17x100mm tube with 5ml appropriate media (most often, since the yeast you grow will lack plasmids, you will use YPDA media). Grow at 30?C o/n, with agitation (the best is to use a culture wheel; if a wheel is not available, hard shaking (300rpm; place tube and some folded paper towels into a beaker and clamp the beaker into the shaker) is a satisfactory alternate.

     reading of an appropriate dilution (1:50 is good for a saturated culture) of the 2. Take an OD600

    o/n culture.

3. Dilute some of the o/n cells into 50ml media total to give a new OD of 0.1. 600

    4. Grow cells for 2-3 hours (for YPDA; if growing yeast with plasmid(s), minimal media is not as “rich” and it will take longer) until OD is between 0.2 and 0.3 (the idea here is that you 600

    want to harvest the cells at early exponential phase).

    5. Spin cells down (5min at 2000rpm in a clinical centrifuge). Remove media. Wash pellet with 5ml water. Spin. Remove water. Wash again. Spin. Remove water.

6. The volumes of solutions that follow are based on the OD reading. If you had an OD 600600

    of 0.27, then add 270ul of ddHO to the cells and transfer the cells/water to a 1.5ml eppendorf 2

    tube. Add 270ul of 0.2M lithium acetate (LiOAc). Use a P1000 and measure the volume of cells, water, and LiOAc. Record.

    7. Gently mix cells, then incubate for 2 hours at 30?C with occasional mixing.

    8. Take the recorded volume from step 6 and divide that number by 5.67. This is the amount of sterile 100% glycerol to add to the competent cells to get final glycerol at 15%. Use a wide-bore tip if available for most accurate pipetting (pipetting glycerol slowly is helpful). Mix well, then aliquot 111ul into 1.5ml eppendorf tubes (from 50ml media, expect 7-8 aliquots). 111ul will be enough for 2 transformations. Immediately place 111ul aliquots into 80?C freezer for

    long-term storage. When using aliquots, do not refreeze after thawing if some cells are left over.

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D. Transformation of competent yeast cells

    This procedure takes about 90 minutes to 2 hours in the class setting. It is possible to transform yeast, obtain colonies, patch them, grow and harvest the yeast (see section E) in one week, though there is not room for “error.” You may prefer to do this 2 weeks before RNA isolation if you unclear about growth rate of your transformed yeast cells (see notes in section E).

Prepare ahead of time:

-- Make yeast cells competent (store at 80?C)

    -- 70% PEG 3350 a good idea to warm bottle/tube up a few hours ahead of time at RT, 70%

    PEG is a solid, but will stay liquid for a few hours at RT, if warmed. The 70% PEG 3350 takes time to initially prepare. MOST of the volume of the solution comes from the PEG, so only a

    O at a time should be added when prepping. A little heat may be applied when little ddH2

    dissolving PEG. When dissolved, the solution should be filter sterilized, which takes time as well due to its great viscosity.

    -- 2 mg/ml sheared salmon sperm DNA (store at 20?C). Set up a boiling water bath for step 1

    below.

    -- 42?C bath

    -- plasmids

    For a 1-plasmid transformation, use 500 ng of each plasmid. For a 2- or 3-plasmid transformation, use 1 ug of each plasmid. This is because the odds of getting plasmids into one yeast cell decrease with number of plasmids.

    1. Boil the 2mg/ml salmon sperm DNA (ssDNA) for 5 minutes; flash cool on ice; leave ssDNA on ice for now.

    2. During boiling, get plasmids from 20?C and competent yeast cells from 80?C. Place on

    ice.

    3. During boiling, ensure your PEG is liquid (VERY quick blast in the microwave is ok if stored in glass bottle; in polypropylene tube, heat in 50?C water bath briefly; cool a bit before use, but do not cool to the point the PEG starts turning white/solidifies). 4. In a final volume of 5ul, combine plasmid(s) and ddHO. Use a 1.5ml eppendorf tube. 2

    5. Add 55ul thawed competent yeast cells to DNA/water.

    6. Add 5ul of the ssDNA (from step 1).

    7. Add 55ul of PEG (70%), light vortex to mix; incubate1 hour at 30?C.

    8. Add 12ul of DMSO (helps open cells to let plasmid(s) in); place at 42?C for 6 minutes. 9. Add 800ul ddHO, mix by inverting tube, centrifuge for 5 minutes at 6000xg, aspirate water, 2

    and resuspend yeast cell pellet in 100ul ddHO. 2

    10. Plate on correct dropout plates.

    11. Grow agar side up at 30?C for 2-3 days.

    12. Using a sterile toothpick, “patch” a few colonies out onto solid media – this can be a source

    of transformed yeast for multiple experiments.

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E. Growing yeast in liquid media for RNA isolation

    The key to a good microarray experiment is to only test one variable at a time. Therefore, if you are looking at, say, WT yeast vs. one of the yeast deletion strains, then the absence of one yeast gene is the variable. Therefore, isolating RNA from yeast harvested at different growth stages would introduce a second variable. Therefore, it is ideal to try and isolate yeast during the same

     near 0.5-0.6). stage of growth (for example, I aim to harvest at mid-exponential phase, at OD600

    Previous published microarray experiments include those that look at what happens when yeast grow during diauxic phase (saturation).

As a rough estimation, for WT yeast in YPDA media, a saturated culture diluted to OD 600

    0.0001-0.0010 late one afternoon will often grow to mid-exponential phase by early the following morning. You should grow yeast ahead of time to learn its growth rate characteristics.

    1. To grow yeast in liquid, during the afternoon of day 1, use a toothpick to sterilely transfer a pinhead amount of yeast from a patch on a solid plate to a 17x100mm tube with 5ml YPDA. Grow at 30?C on wheel (or with agitation in shaker see Section C step 1), o/n.

2. During the morning of day 2, take OD readings. Do the same in late afternoon. This 600

    will help you get an idea of growth rate.

    3. Using the afternoon readings, subculture the yeast with the goal that in the morning, you will be able to harvest 8ml yeast in mid-exponential phase (OD = 0.5-0.6). The degree of 600

    subculturing varies from yeast strain to yeast strain and if the yeast are being grown in YPDA or minimal media. As noted in the prelude to this section, it is challenging to immediately know what is the volume to subculture down to. Even after you have a good idea about growth rates of your yeast, I suggest you subculture each sample to three different ODs so that you have a “range” when you check OD’s on the morning of day 3 (step 4). Note that at this point I make

    duplicates for each subculture point, so that I can have a pair of yeast pellets for each group of students. This way if there is a problem with one sample during the RNA extraction, there is a possible back-up. As an example of subculturing, if my OD reading at 4pm was 4.5, and I 600

    wanted to subculture to 8ml at OD 0.001, then the math is 8mlx(0.001/4.5) = 0.00178ml = 600

    1.78ul of saturated culture into 8ml media.

4. Measure OD’s of all samples in the morning (day 3 now); select samples in mid-600

    exponential range. Spin 5 minutes at 2000rpm in clinical centrifuge. Remove media. Wash pellets with 5ml ddHO. Spin. Remove water. Wash pellets again with 5ml ddHO. Spin. 22

    Remove water. Add 1ml ddHO, transfer water/cells to a sterile (autoclaved) 1.5ml eppendorf 2

    tube (a “safe-lock” tube if you have them, such tubes are nice to have when working with phenol in section F, below), spin 3 minutes at 6000xg, remove water, store pellets at 80?C until needed

    (can store for several days).

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F. Isolation of RNA from yeast using heated, acid buffered phenol

    This method, which can be done in one three-hour lab period, is robust; you can repeatedly obtain 75-100ul of ca. 1-1.5mg/ml RNA from 8ml cells at OD600=0.5 (4 OD cells). It is a protocol I have used in my microbiology lab course, and hundreds of times in the research lab.

    I do not keep the phenol in a hood; the bottle can remain closed for most of the time, and it is easier to pipet out of a bottle on a benchtop instead of standing outside of a hood. I do only let one student at a time near the phenol “workstation” – a heated stirplate and I supervise removal

    of phenol.

    REMINDER/CAUTION: The most critical reagent in this procedure is heated, acidic phenol. In general, any phenol is EXTREMELY DANGEROUS and even 1 microliter on exposed skin will cause a severe burn. Phenol can also “eat through” clothing and cause burns. In addition, the phenol we use today will be heated to 65?C.

IF YOU HAPPEN TO SPILL PHENOL ON YOU OR SOMEONE ELSE: IMMEDIATELY

    run cool water over the affected area to dilute the phenol. Carefully remove clothing if necessary. By working with only a fraction of a milliliter at a time, a large spill should not be a concern.

    Note that all solutions used throughout the remainder of this protocol are made up with RNase free water. Ideally, access to a Milli-Q system with a cartridge that removes RNases from water can be used as a source of water. If unavailable, as this is expensive to buy and maintain, consider buying non-DEPC treated RNase free water from Ambion. DEPC is very toxic and is not ideal to work with. The protocol below has not been tried with DEPC treated water used to make solutions/suspend the final RNA pellet, and I cannot guarantee that use of DEPC water will be ok here.

    You should consider using “new” boxes of pipet tips that can be marked as “RNA only.” You also may want to consider using filtered/barrier tips if you are concerned that the interior of the pipetters are not “clean.”

    (The Section F protocol could be copied and pasted into a document that can be given to the students to perform this part of the experiment. Feel free to borrow this without my permission, but I would appreciate this document being referenced.)

    1. Ensure that there is a water bath warmed to 65?C. The Instructor will have phenol buffered with “Buffer A” (50mM sodium acetate pH 5.2, 10mM EDTA, 1% SDS) warming in the bath. This buffered “hot phenol” will have been made up ahead of time by the Instructor by mixing

    equal volumes of Buffer A with melted crystalline phenol (as well as 0.1g 8-hydroxyquinolone per 100ml phenol; see step 4 below), stirring, letting the layers separate, withdrawing the upper non-phenol layer, and repeating 2 more times. At the end, about 25ml of Buffer A was left on top of the phenol layer, but by heating and stirring, these layers will be mixed when you access the phenol. Thus, the phenol is “saturated” with Buffer A.

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2. Put on gloves. Wear gloves throughout the procedure. Obtain 1.5ml “safe-lock” eppendorf

    tubes with a frozen (-80?C) yeast cell pellet from your Instructor and place them on ice for about 5 minutes to thaw. (You will process two “identical” pellets this week – one serving as a

    backup in case of accidental spilling). These yeast cell pellets are the end-product of one yeast colony harboring plasmids grown in 8ml of liquid media to mid-exponential phase, spun, then

    O to remove media. washed two times with ddH2

    3. You will be given a small aliquot of Buffer A. Using your P1000 pipetter with an appropriate sized barrier/filter tip, resuspend each yeast cell pellet carefully in 300ul Buffer A. Eject this tip, as well as all tips used today, in your waste container these tips are biohazards

    and need to be autoclaved by the Instructor after lab.

    4. Note that the hot phenol appears cloudy/milky, with a light yellow tint. The yellow is a color indicator, 8-hydroxyquinolone, which allows us to easily visualize the phenol layer. This will be important momentarily. The phenol will have been prewarmed to 65?C but you will find it on a heated sitrplate the acid phenol will be gently stirring it needs to be mixed when you

    remove your aliquot. Carefully pipet 600ul of the hot phenol into the eppendorf tubes with the recently resuspended yeast cells. Close the lid of the tubes securely and vortex vigorously for 30 seconds to begin to lyse the cells; the phenol is used to separate the nucleic acids from protein the protein will be found with the phenol after centrifugation; the nucleic acids will be in the upper, aqueous (“milky”) phase – see step 7, for example.

    5. Incubate tube in floatation device in 65?C water bath (see step 1) for 5 minutes. Take care to avoid knocking over the phenol bottle if it is currently also in the bath. Invert the tubes a few times during the incubation period to thoroughly mix the cells/Buffer A/hot phenol.

6. Continue to work in groups and try to share the microcentrifuge balance your tubes! Spin

    at 15,000xg for 5 minutes.

    7. This is a little tricky. After the spin you should see a yeast pellet at the bottom of the tubes, then yellow phase (phenol), and then a milky white phase on top. There may be a thin whitish-goo layer between the milky Buffer A layer and the phenol. Use a P200 and carefully remove

    the yellow (lower) phase. Pipet out the yellow phase into a small waste container (such as a 2ml eppendorf tube). DO NOT pour this waste into a wastebasket or sink the liquid phenol waste

    will be disposed by the Instructor in a special manner. Note that there should be 400-700ul of the yellow layer (do not disturb the cell pellet), so for each tube you will need to repeat the withdrawals 2-4 times. For this step, leaving behind a small amount of phenol layer is ok, and preferable to removing some of the cells or upper milky layer. Do take extreme care not to touch the pellet with the pipet tip when removing the lower yellow phenol phase!!!

    8. Pipet 600ul of the mixed, hot phenol to the leftover cell pellet/milky layer (upper phase). Heat at 65?C for about 1 minute, then vortex for 30-60 seconds. During vortexing, stop occasionally and see if the pellets break up (hold tube to light and invert). Goal: disperse the cell pellets.

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    9. Repeat steps 5 and 6. During the 5 minute spin (step 6 repeat), add 50ul of 3M sodium acetate pH 5.5 to each of two new “safe-lock” 1.5 ml eppendorf tubes.

10. After the spin of step 9 (the “step 6 repeat”) is done, use a P200 to transfer the upper

    aqueous (milky) phase (ca. 700ul) to a tube with the 3M sodium acetate. Place the tubes with the yeast cell pellet and acid phenol to the side so that the instructor can dispose of them. During the transfer using the P200, taking a little phenol layer with you is ok here, and is better than leaving some milky phase behind. Do try to avoid the really gooey white material that may accumulate at the layer interface, though. Make sure you keep track of your two samples by proper labeling of tubes!

    11. Add 500ul of refrigerated Tris buffered phenol:chloroform (1:1) to each of your two eppendorf tubes with the milky upper layer/sodium acetate from step 10. There will be a top and bottom layer of the phenol/chloroform unlike the acid phenol, which is used as a mixed

    suspension, do not mix the phenol:chloroform, and do not take the upper layer -- ensure you take

    liquid from the bottom phenol layer by verifying the yellow color (due to addition of hydroxyquinolone upon preparation). NOTE: although this is not acid phenol, but just a mix of phenol:chloroform, still take care not to spill the phenol:chloroform also can cause severe

    burns.

    12. Vortex the tubes with the phenol/chloroform combined with the aqueous layer for 30 seconds, then place the tubes in the eppendorf tube mixer for 3 minutes. Then spin the tube in the microcentrifuge (again, share the centrifuge and balance the tubes!) for 3 minutes at 15,000xg.

    13. During the spin, add 1ml of 100% ethanol to two new eppendorf tubes. After the spin is complete, use a P200 to transfer each upper aqueous layer (again, you will need to do 3-4 transfers) to the appropriately labeled tube with ethanol. Note that after the spin, the upper aqueous layer should be totally clear. Also note, that you should transfer only the upper aqueous layer and not the interface or yellow phenol layer. Better to leave a little upper aqueous layer behind than to contaminate with a miniscule amount of lower layer.

14. Invert tubes to mix (do not vortex) and place them in a 80?C freezer. Chill 20 minutes.

15. Spin tubes 5 minutes at 15,000xg. Place the hinge of the tube lid towards the “outside” of

    the rotor your RNA should pellet on the outside/back wall at the bottom of the tubes.

    Remove supernatant by pipetting the liquid out of the tube (using a P200). To avoid touching

    the pellet, when pipetting up liquid, glide the pipet tip down the “inside/front” wall of the eppendorf tube. It can be helpful to do a quick “pulse” spin in the microcentrifuge before trying to remove the last few microliters of ethanol out of each tube.

    O. Vortex 30 seconds; then place the tubes in the eppendorf tube mixer 16. Add 300ul ddH2

    for 3 minutes to dissolve RNA (alternatively, use a standard vortexer set on medium for mixing, but one must vortex for 3 minutes).

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    17. To each of the two eppendorf tubes from step 16 add 30ul 3M sodium acetate pH 5.5 and 900ul 100% ethanol. Mix by inversion (not vortexing). Repeat steps 14 and 15.

    18. Rinse each pellet with 300ul ice-cold 70% ethanol. Spin 2 minutes in microcentrifuge. Remove supernatant using a P200 as described above. Pulse spin and NOW USE a P20 to

    remove the last few microliters of ethanol. Air dry 5-10 minutes (leave tube open on benchtop

    but be careful to not send dust or anything over the tube while drying keep your RNA free

    from RNAses!

    19. Make sure there is no liquid alcohol in the tube (this is critical!), then suspend each pellet in

    O. Vortex 30 seconds, then place tubes in the eppendorf tube mixer for 3 minutes. 75ul ddH2

    Then store your RNA at 80?C until next lab period.

If desired, you could spend part of a lab period taking ODreadings to calculate RNA 260/280

    concentration and determine purity (ODratio between 1.8-2.0, ideally 2.0). This choice 260/280

    depends on time. In the past, I have taken ODs myself and asked the students to determine how much RNA they isolated (concentrations are needed for prepping cDNA, the next step).

    G. Preparation of cDNA from total yeast RNA using components from the Genisphere 900 kit (recall note preceding Section A about obtaining reverse transcriptase).

    Note that from here forward, this protocol is assembled from Genisphere’s instructions based on yeast microarrays of 70-mers. It is strongly recommended that you also read Genisphere’s

    instructions upon preparing to do the array work.

    Section G can be easily done in a 3 hour lab period. There is a 2 hour incubation, which can be used as time for discussion, working on MAGICTool sample files, etc.

    Note that this procedure uses VERY small volumes. Having P2 or P10 pipets here would be VERY helpful but are not absolutely required. Ensure that small volumes (less than 2ul) are visibly transferred to tubes.

    Read ahead and get water baths, etc. ready. You will need baths at 42?C, 65?C, and 80?C. Alternatively, you can start with baths at 42?C and 80?C, then cool the 80?C down to 65?C during the 2 hour cDNA synthesis (step 6).

    Here, for simplicity, we are going to do a microarray where the WT/control sample will be called “sample A” and the experimental sample will be called “sample B.” Here, sample A will be ultimately be labeled with Cy3 (green) and sample B with Cy5 (red). Consider doing a “dye reversal” as well on a separate array, with sample A/Cy5 and sample B/Cy3. Again, for

    simplicity, we will deal with sample A/Cy3 and sample B/Cy5 here in this protocol.

    1. Put on gloves. Get two 1.5ml eppendorf tubes for the next step. Label one “A” and the other “B.”

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