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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%.

    3

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.

    4

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.