Protocols

Mechanical disruption of yeast cells

1.Grow yeast cells to the desired growth rate. A OD₆₀₀ of 0.5 is recommended for best results of cell lysis, but higher densities are also possible.

2. Harvest the cells by centrifugation at 3000rpm for 3 min.

3. Resuspend in a very small volume of growth medium.

4. The suspension is sucked into a pipette and released as individuel drops directly into liquid nitrogen.

5. Frozen cell drops can be stored at -80°C.

6. Pre-cool a 5ml Teflon vessel in liquid nitrogen (and the little spoon).

7. Add a 7 mm bead made of tungsten carbide and frozen cells (two or three frozen drops of cells) équivalent to about 15 OD₆₀₀ units of cells.

8. Close the flask with the precooled cap and place it into the holder of the microDismembrator (Braun, Melsungen)

9. Set the shaking frequency to 2600 rpm and the opération time to 2 min and start.

RNeasy Mini Protocol for Isolation of Total RNA from Yeast

Mechanical Disruption Protocol

Use an appropriate number of cells

Important notes before starting

- For RNA isolation from yeast, cells should be harvested in log-phase growth. Cell pellets can be stored at -80°C for later use or used directly in the procedure. Cell lysates (in Buffer RLT, step 5) can be stored at -80°C for several months. To process frozen lysates, thaw samples for 15-20 min at 37°C in a water bath to dissolve salts. Continue with step 6.

- b-Mercaptoethanol (b-ME) must be added to Buffer RLT before use. b-ME is toxic; dispense in a fume hood and wear appropriate protective clothing. Add 10 µl b -ME per 1 ml Buffer RLT.

- Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96-100%), as indicated on the bottle, to obtain a working solution.

- Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and then place at room temperature.

- Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. Guanidine is an irritant. Take appropriate safety measures and wear gloves when handling.

The ARN purification begin at the step 3 of the qiagen protocol after the mecanical disruption with the microDismembrator

3. Transfert the cell fragment in an eppendorf containing 600µl Buffer RLT, and vortex to resuspend the cell pellet. Note: Ensure that b-ME is added to Buffer RLT before use (see "Important notes before starting").

6. Add volume (usually 400 µl) of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge.

If some lysate is lost during homogenization, adjust volume of ethanol accordingly. Note: Visible precipitates may form after the addition of ethanol, but this will not affect the RNeasy procedure.

7. Apply up to 700 µl of the sample, including any precipitate that may have formed, to an RNeasy mini column placed in a 2 ml collection tube (supplied). Close the tube gently, and centrifuge for 15 s at =8000 x g (=10,000 rpm). Discard the flowthrough.*

Reuse the collection tube in step 8.

If the volume exceeds 700 µl, load aliquots successively onto the RNeasy column, and centrifuge as above. Discard the flow-through after each centrifugation step.*

8. Add 700 µl Buffer RW1 to the RNeasy column. Close the tube gently, and centrifuge for 15 s at =8000 x g (=10,000 rpm) to wash the column. Discard the flow-through and collection tube.*

9. Transfer the RNeasy column into a new 2 ml collection tube (supplied). Pipet 500 µl Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 15 s at =8000 x g (=10,000 rpm) to wash the column. Discard the flow-through. Reuse the collection tube in step 10.

Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see "Important notes before starting").

10. Add another 500 µl Buffer RPE to the RNeasy column. Close the tube gently, and centrifuge for 2 min at =8000 x g (=10,000 rpm) to dry the RNeasy silica-gel membrane. Continue directly with step 11, or, to eliminate any chance of possible Buffer RPE carryover, continue first with step 10a.

It is important to dry the RNeasy silica-gel membrane since residual ethanol may interfere with downstream reactions. This centrifugation ensures that no ethanol is carried over during elution.

Note: Following the centrifugation, remove the RNeasy mini column from the collection tube carefully so the column does not contact the flow-through as this will result in carryover of ethanol.

10a. Optional: Place the RNeasy column in a new 2 ml collection tube (not supplied), and discard the old collection tube with the flow-through. Centrifuge in a microcentrifuge at full speed for 1 min.

11. To elute, transfer the RNeasy column to a new 1.5 ml collection tube (supplied). Pipet 30-50 µl RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube gently, and centrifuge for 1 min at =8000 x g (=10,000 rpm) to elute.

12. If the expected RNA yield is >30 µg, repeat the elution step (step 11) as described with a second volume of RNase-free water. Elute into the same collection tube. To obtain a higher total RNA concentration, this second elution step may be performed by using the first eluate (from step 11). The yield will be 15-30% less than the yield obtained using a second volume of RNase-free water, but the final concentration will be higher.

Probe preparation for membranes

Reverse transcription :

2. Add 1 µl oligo dT₁₅ (500 ng, Life Technologies).

3. Vortex, mix and do a pulse of centrifuge

4. Incubate at 70°C for 10 minutes. Cool briefly on ice.

5. Place at 42°C.

6. Mix and add: 6 µl 5xFirst Strand Buffer
3 µl 0.1M DTT
1.5 µl  AGT+C mix
10.5µl final volume 7. Add 5µl ³³P a-dCTP (50 µCi, Amersham Pharmicia, AH9905)

8. Add 2 µl SuperScript RT (200U, Life Technologies)        ->don't forget 30 µl final volume

9. Incubate at 42°C for 30 min and add 1 µl SuperScript RT (200U)

10. Incubate one more time for 30 min at 42°C

11. Before opening the tube do a pulse of centrifuge because of the water on the lid

12. Take out 1 µl to check the incorporation of radioactivity. See protocol for "Precipitation of nucleic acids with TCA".

Buffers and solutions

• DEPC-treated H₂0 (Eau en bas)
• Oligo dT₁₅ (500 ng/ µl)
• [a-33P]-dCTP (2500 Ci/mmol) (Amersham)
• SuperScript RT (200U/lµl) (Gibco BRL)
• 5x First Strand Buffer (Gibco BRL): 250 mM Tris/HCI pH 8.3
• 375 mM KCI
• 15 mM MgCl₂
• 0.1M DTT (Gibco BRL)
• AGT+Cmix 16 mM dATP
• 16 mM dGTP
• 16 mM dTTP
• 100 µM dCTP

Precipitation of nucleic acids with TCA

2. Mix it with 7 µl 0.05 M EDTA pH 8, 7 µl H₂O and 5 µl tRNA (10mg/ml) 3. Put 9.5 µl of aliquots on the centre of two separate pieces of GF-F Whathmann paper. Mark them differently for instance by cutting the corners in various way. One filter is kept for measuring the total amount of radioactivity (total filter). The other is to measure only the acid-precipitate radioactivity (TCA filter). Under these conditions, DNA and RNA molecules more than 50 nucleotides will be precipitated on the surface of the filter.

4. Let them dry completely at room temperature

5. Soak the TCA filters in a beaker containing ice-cold 10% TCA and 1% sodium pyrophosphate for 5 minutes. Swirl the beaker from time to time.

6. Discard the liquid and repeat twice.

7. Wash the filters in 96% ethanol at room temperature for two minutes

8. Let them dry completely.

9. Insert each of the filters into a scintillation vial and 3 ml of scintillation liquid for solid samples, to enhance the measurement of the radioactivity.

10. Measure the ³³P with an appropriate program of the liquid scintillation counter.

11. % of incorporation = counts on "TCA filter" / counts on "total filter"

Alkaline hydrolysis of RNA

1 µl 0.5M EDTA pH8.0

3 µl 3 M NAOH

2. Incubate at 65°C at least 30 minutes.

3. Incubate at room temperature for 15 minutes.

4. Add 10 µl 1M Tris/HCl pH 8.0

3 µl 2N HCl

Buffers and solutions:

• 1 % SDS
• 0.5M EDTA pH 8.0
• 3 M NAOH
• 1 M Tris/HCI pH 8.0
• 2 N HCl

Isopropanol precipitation

5 µl tRNA 10 mg/ml

60 µl isopropanol

2. Precipitate at -20°C for 30 minutes (in a radioactive box)

3. Centrifuge at maximum speed for 30 minutes in the hotlab (note the ratio of radio activity between the pellet abd the supernatant)

4. Redissolve the cDNA in 100µl ddH₂0. It is possible to take out 5 µl of cDNA to control later on an alkaline electrophoresis the quality of the total cDNA

5. (Alliteratively use a QlAquick Nucleotide Removal Kit.)

Buffers and solutions:

• 3 M NaOAc pH 5.3
• tRNA (10 mg/ml)
• isopropanol

Probe preparation for slides

ChipShot Direct labelling (Promega)

Le protocole est divisé en deux parties :
ChipShot Direct labelling TOTAL RNA (Etape 1 à 4)
ChipShot Membrane Clean-up system (Stage 1 to 10)
Voir le protocole (.pdf)

Label star labelling (Qiagen)

This is the standard protocol for direct labeling of cDNA with biotin-dCTP, cyanine 3-dCTP, or cyanine 5-dCTP, using 0.2-50 µg RNA. The amount of RNA corresponds to the total amount of RNA present, including any rRNA, mRNA, viral RNA, or carrier RNA present.

Important notes before starting - Set up all reactions on ice to avoid premature cDNA synthesis and minimize the risk of RNA degradation.

- Be sure that everything is mixed well by vortexing.

- Ensure that this protocol is suitable for the modified nucleotide that you are using (see Table 2, page 12 on qiagen protocol).

- The protocol is optimized for use with 0.2-50 µg total RNA or 0.2-5 µg mRNA. For amounts >50 µg total RNA, scale up the reaction linearly to the appropriate volume (see Table 4, page 15 on qiagen protocol).

- The protocol is optimized using oligo-dT primers. If you intend to use other primers, please refer to Appendix D on qiagen protocol

A) Preparation of dNTP Mix C

Note: The total volume of dNTP Mix C (64 µl or 260 µl) is sufficient for 12 or 50 identical labeling reactions respectively (depending on kit size). If using different cDNA labelling protocols with the LabelStar Array Kit ensure that appropriate aliquots of dNTP Mix C are prepared. After use, store the remaining dNTP Mix C in the LabelStar cDNA Labeling Module (box 1 of 2) at -20°C.

1. Thaw dATP, dCTP, dTTP, and dGTP solution on ice. Mix each solution, for example, by vortexing. Centrifuge briefly and keep on ice.

2. Prepare the dNTP Mix C (Table 3) on ice in the tube provided. Mix and keep the tube in ice

Table 3. Preparation of dNTP Mix C using cyanine 3-dCTP, or cyanine 5-dCTP

Be sure that everything is mixed well by vortexing.

B) Protocol for cDNA labeling

1. Thaw the template RNA solution, LabelStar Reverse Transcriptase, dNTP Mix C (see Table 3), RNase inhibitor, and labeled dCTP (1 mM) on ice. All other components, oligo-dT primer solution, 10x Buffer RT, Denaturation Solution Plus, and RNase-free water should be thawed at room temperature, mixed, and kept on ice.

Note: cyanine 3-, and cyanine 5-labeled dCTP (1 mM) are not provided with this kit.

Note: dNTPs and labeled nucleotides are labile reagents. Freeze nucleotides immediately.

2. Adjust the volume of your RNA solution to a total volume of 18 µl with RNase-free water.

3. Add 2 µl Denaturation Solution Plus to the 18 µl RNA solution. Mix and centrifuge briefly.

4. Incubate at 65°C for 5 min and cool down on ice immediately. Centrifuge briefly.

5. Prepare a fresh master mix on ice according to Table 4. Mix thoroughly and carefully by vortexing. Centrifuge briefly and store on ice.

The master mix contains everything except the denatured template RNA.

Note: Be sure to mix the master mix throughly before adding the denatured template RNA.

6. Add 20 µl of denatured template RNA (step 3 and 4) to the individual tubes containing the master mix. Mix and centrifuge briefly.

Note: Be sure that everything is mixed well by vortexing.

7. Incubate for 120 min at 37°C.

8. Add 2 µl of Stop Solution LS to the individual tubes. Mix and centrifuge briefly.

Note: RNA degradation step is not needed.

9. Proceed to the purification protocol.

For optimal sensitivity and background, use the LabelStar Cleanup Module (box 2 of 2). If purification will not be performed immediately, store tubes at -20�C.

Labelling first-strand cDNA with Cy3- or Cy5-nucleotides

CyScribe First-Strand cDNA Labelling Kit from amersham pharmacia biotech for preparation of Cy3- and Cy5-labelled cDNA for Microarray Hybridization

Ref: RPN 6202

- Set a water bath at 70 ºC and another at 42 ºC.

- Place the required reagents from the kit, excluding the enzyme on ice to thaw. Only place the enzyme on ice immediately prior to use. Make sure that the contents of all tubes are thoroughly thawed and mixed before pipetting solutions from them.

- To anneal primers to mRNA, add the following labelling reaction components to a 1.5 ml amber microcentrifuge tube on ice:

mRNA, 1 µg X µl or RNA total 20 µg

Random nonamers 1 µl

Anchored oligo(dT) 1 µl

Water (supplied) Y µl

Total 11 µl

The volumes corresponding to x and y should be adjusted so that the total reaction volume is 11 µl.

- Mix gently by pipetting up and down.

- Incubate the reaction mixture at 70 ºC for 5 minutes.

- Let the reaction mixture cool at room temperature for 10 minutes to allow the primers to anneal with the mRNA template.

- Spin down the reaction mixtures for 30 sec in a microcentrifuge to collect all reaction components at the bottom of the tube.

- Place the annealed reaction mixture on ice and add the labelling components in the following order:

5 x CyScript buffer 4 µl

0.1 M DTT 2 µl

dUTP or dCTP nucleotide mix 1 µl

dUTP or dCTP CyDye-labelled

nucleotide 1 µl

CyScript reverse transcriptase 1 µl

Total volume 20 µl

Remove the CyScript enzyme from freezer just before removing aliquots into labelling reactions and immediately afterwards return the enzyme to a freezer at -15 ºC to -30 ºC.

Use the dCTP nucleotide mix in conjunction with either Cy3-dCTP or Cy5-dCTP. Likewise, use dUTP nucleotide mix either with Cy3-dUTP or Cy5-dUTP. These nucleotide mixtures have been optimized for use with their corresponding CyDye-nucleotide and are not interchangeable.

- Mix the reactions by vortexing and spin them for 30 sec in a microcentrifuge.

- Incubate the reactions at 42 ºC for 1.5 hours.

- Store the labelled cDNA on ice for immediate purification or place at -15 ºC to -30 ºC for storage. Protect from light and do not store in a frost-free freezer.

Purification of labelled cDNA: two techniques are available; one uses columns and the other uses the isopropanol precipitation

It is necessary to remove mRNA from the single-stranded cDNA to promote hybridization of the cDNA probe to immobilized microarray targets and to minimize hybridization with complementary mRNA in solution. Removal of unincorporated CyDye-nucleotides is also necessary for minimising hybridization background and for improving the sensitivity of detection of low abundance targets. The removal of mRNA can be achieved in two steps. First mRNA is degraded into short oligomers with alkaline treatment. Then these short oligomers, as well as unincorporated nucleotides, are removed with spin column chromatography. Amersham Pharmacia Biotech recommend the use of AutoSeq G-50 spin columns for this purpose. These columns have been found to remove up to 99.9% of free nucleotides and result in consistent recovery of labelled cDNA. Some loss of material will occur during the purification procedure. The reagents for removal of mRNA and purification of labelled cDNA are not included in the CyScribe First-Strand cDNA Labelling Kit.

Technique 1: with AutoSeq G-50 columns

Degradation of mRNA

- Adjust a waterbath to 37 ºC.

- Add 2 µl of 2.5 M NaOH into each microcentrifuge tube containing labelling reactions.

- Mix the reaction mixtures by vortexing and spin them for 30 sec in a microcentrifuge.

- Incubate the samples at 37 ºC for 15 minutes.

- Add 10 µl of 2 M HEPES free acid to each reaction tube.

- Mix the reaction mixtures by vortexing to ensure that all of the contents are neutralized and spin them for 30 sec in a microcentrifuge.

- The labelling reactions are now ready for purification or can be stored at -15 ºC to -30 ºC.

Removal of nucleotides and short oligomers with AutoSeq G-50 columns

AutoSeq G-50 columns contain Sephadex G-50 DNA grade F and are supplied pre-equilibrated in water containing 0.05% Kathon cG/ICP Biocide as preservative.

Prepare the columns for nucleotide removal as follows:

- Resuspend the resin in the column by vortexing gently.

- Loosen the cap a quarter of a turn and snap off the bottom closure.

- Place the column in a 1.5 ml screw-cap microcentrifuge tube for support. Alternatively, remove the cap from a standard microcentrifuge tube and use this tube for support. If using a 1.5 ml microcentrifuge tube, 10-20 µl of fluid will remain in the tip of the column after spinning. Blot this fluid from the column using a clean paper towel before applying sample into the column.

- Spin the column for 1 minute at 2000 ×g. Start the timer and the microcentrifuge simultaneously. Use the column immediately after preparation to avoid drying of the matrix.

Purify the labelled cDNA as follows:

- Place the column in a new 1.5 ml tube and slowly apply the sample to the centre of the angled surface of the compacted resin bed, being careful not to disturb the resin. Do not allow any of the liquid to flow around the sides of the bed.

- Spin the column for 1 minute at 2000 ×g. Start the timer and the microcentrifuge simultaneously. The purified sample is collected at the bottom of the column. Discard the column.

- The purified cDNA is ready for use in microarray hybridizations or can be stored at -15 ºC to -30 ºC protected from light.

Technique 2: Isopropanol precipitation

- Add to the 20 µl labelled cDNA

2.5 µl 50mM EDTA (pH8.0)

1 µl 10N NaOH

incubate 65°C 20 min

add 2 µl 5M acetic acid

1vol isopropanol (25µl)

incubate at -20°C 20 min

centrifuge 5 min 4°C

rinse the pellet with 70% ethanol

quick spin 4°C

add to the pellet 5µl DEPC-treated water

- The purified cDNA is ready for use in microarray hybridizations or can be stored at -15 ºC to -30 ºC protected from light.

Technique 3: Purification of labeled cDNA with Labelstar clean up module

Protocol for Purification of Labeled cDNA

Using a Microcentrifuge

Important notes before starting

- Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).

-All centrifugation steps are carried out at >10,000 x g (13,000 rpm) in a conventional tabletop centrifuge.

-Ensure that reactions are mixed well by vortexing.

-For users performing dual-color analysis: pooling of dual-color labeled cDNA prior to purification is possible if the amount of RNA used per individual reaction was less than 25 µg. If >25 µg RNA is used per reaction, purify dual-color labeled cDNAs separately using two MinElute spin columns.

-Take appropriate safety measures when working with radioactive isotopes.

1. Add 260 µl Buffer PB to the total cDNA labeling reaction and mix by gentle vortexing. Note: Add 520 µl if two reactions are pooled for purification.

2. Place a MinElute spin column in a 2 ml collection tube.

3. To bind cDNA, apply the sample to the MinElute spin column and centrifuge for 1 min.

4. Discard the flow-through fraction and replace the MinElute spin column in the same 2 ml collection tube.

IMPORTANT: If using radioactively labeled modified nucleotides, use a new tube for each step and dispose of radioactive waste according to your institution's safety regulations.


5. To wash, add 0.75 ml Buffer LS to the MinElute spin column and centrifuge for 1 min. Note: If cleaning up radioactively labeled cDNA, use 0.7 ml Buffer LS.

6. Discard the flow-through and place the MinElute spin column back in the same 2 ml collection tube.

7. To wash, add 0.75 ml Buffer PE to the MinElute spin column and centrifuge for 1 min. Note: If cleaning up radioactively labeled cDNA, use 0.7 ml Buffer PE.

Note: Buffer PE is supplied as a concentrate. Ensure that ethanol is added before use (see "Important notes before starting").

8. Discard the flow-through. Place the MinElute spin column back in the same 2 ml collection tube and centrifuge for an additional 1 min at maximum speed.

9. Place the MinElute spin column in a clean 1.5 ml microcentrifuge tube.

10. To elute the cDNA, add 10 µl Buffer EB (10 mM TrisµCl, pH 8.5) to the center of the MinElute spin column membrane. Wait for 1 min, and centrifuge the column for 1 min.

Note: Ensure that the elution buffer is dispensed directly onto the MinElute spin column membrane for complete elution of bound cDNA. The average eluate volume is 9 µl from 10 µl elution buffer.

IMPORTANT: Buffer EB should be used for elution in all cases, including those where dye-coupling to aminoallyl-cDNA will be performed after purification.

11. Optional: Repeat the elution with a further 10 µl elution buffer. If labeling was performed using >25 µg RNA, this second elution step is recommended for increased recovery.

12. Proceed with hybridization of labeled cDNA to the array or with dye-coupling using NHS-esters (if aminoallyl-dUTP was used).

-Note: Purified labeled cDNA is ready for hybridization to arrays. Proceed with a typical hybridization protocol. If necessary, pool cDNAs that were labeled with cyanine-3 and cyanine-5. Precipitation or drying of labeled cDNA may be necessary to obtain a smaller volume prior to hybridization. Mix labeled cDNA with hybridization solution and follow your standard procedure.

-After synthesis and purification of the cyanine-labeled target cDNA, measure absorbance at 260, 550, and 650 nm. Best hybridisation results are obtained with cDNA having a frequency of incorporation (FOI) of at least 20 labeled nucleotides per thousand.

-Using cDNA of lower FOI reduces the sensitivity of the assay. An FOI greater than 50 is indicative of incomplete removal of unincorporated labeled nucleotides. Determine the yield and label strength of target cDNA using the following formulae:

Amount of target cDNA (ng) = A260 x 37 x total volume of cDNA (µL)

Labeled nucleotides incorporated (pmoles) =

for Cy3: A550 X total volume of cDNA/0.15

for Cy5: A650 X total volume of cDNA/0.25

-FOI = Labeled nucleotides incorporated x 324.5/ amount of target cDNA

Note: These equations were generated using the following constants:

One A260 unit of single-stranded DNA = 37 µg/ml;

extinction coefficient of Cy3 = 150,000 M-1cm-1 at 550 nm;

extinction coefficient of Cy5 = 250,000 M-1cm-1 at 650 nm;

average molar mass of dNTP = 324.5.

Hybridisation for membranes

Hybridisation with complex probe :

1. Pre-hybridise the filter for 2 hours at 65°C with church buffer.

2. Denature the probe by adding 1/10 volume 3 M NAOH (or by incubating 5 minutes at 100°C).

3. Hybridise overnight at 65°C with the probe mixed in 5 ml pre-heated church buffer during 19 hours.

Buffers and solutions:

• Church buffer : 0,5 M Na-Phosphate pH=7.2
• 7% SDS
• 1 mM EDTA

Washing the filter

2. Wash 30 minutes at 65 °C with the washing buffer.

Washing buffers

• 40 mM Na-Phosphate pH=7.2
• 0.1 % SDS

Exposure

1. Drain the membran but avoid drying.

2. Wrap the film in saran wrap carefully, make sure that the wrap is as flat as possible (avoid air bubbles since they will decrease the signal).

3. Expose for 1-3 days on an imaging plate (IP).

Regeneration of the filter :

1. Boil 300 ml of the stripping buffer in a micro-wave.

2. Pour 100 ml of the solution directly onto the filter (repeat).

3. Pour the rest onto the filter and allow to cool to room température (25°C).

4. Dry the filter on a whathman paper.

5. Expose the filter on an IP to check the regeneration

Stripping buffer

• 5 mM Sodium phosphate buffer pH 7-7.5
• 0. 1 % SDS

Light deshybridation method
SOUTHERNS, NORTHERNS, MICROARRAYS

A. SPECIFIC MATERIEL :

Solutions 2, 3, 4 are stored at laboratory temperature .

1. 1 nucl�oside a-thiotriphosphate.

If dATP :

If dCTP:

2.Medicinal iodine
     Composition, for 100 grammes : Iode, 5 g ; KI, 3 g, ethanol 95µ, 85 g ; H20, 7 g.

3. Buffer Tris 1,5 M, pH 8,8 (solution for gels Laemli)

4. Thiocyanate de sodium 1M

B. METHOD

B1. Make labelling as usually and substitute dCTP for dCTPaS if dATP 33P or substitute dATP for dATPaS if labelling dCTP 33P.
First option offer the advantage of spliting close to C and so for shorter oligos GC-rich .
Make hybridization and wash as usually.

B2. Wash the gel during 30 min in 0,2% SDS 80µ

B3. Put at laboratory temperature, eliminate SDS, replace by distillated water in excess.

B4. Prepare just before use it 50 ml of this solution

B5. Eliminate distillated water and incubate filter with low roughness at laboratory temperature during 10 minutes in solution B3. Filter must become uniformly orange.

B6. Prepare 50 ml of dilution of reduction :

Eliminate solution B3, replace by the dilution of reduction, Put with low roughness during 10 min at 68°C.
Filter must be very white.

B7. Eliminate the dilution de reduction. Filter is ready for a new hybridization or can be enveloped in Saran and stored at -20°C.

COMMENTS :

1. 1 micromole of dNTP aS is enough for 4 ml of labelling reaction (concentration of 250 µM) for at less 100 hybridizations.
2. Deshybridization is at less 95%, if sequences are rare for the le nucleotide aS, exceptionally, deshybridization can be worse
3. Be careful ! iode task clothes irreversibely and some person are allergic.

UltraGAPS Coated Slides - Instruction Manual

Preparation and hybridization of DNA Microarrays

General Considerations :

Composition of spotting solution.

The chemical and physical properties of the spotting solution greatly influence DNA retention, spot morphology, and hybridization efficiency. Corning recommends the use of the Pronto! Universal Spotting Solution (Cat. No. 40019, 40025, and 40027) because of its low evaporation rate and nuclease-inhibiting properties. Solvent evaporation causes the concentration of DNA and other nonvolatile components of the spotting solution to rise, leading to time-dependent changes in spot quality, suboptimal array uniformity, and the eventual loss of the spotting solution. The most commonly used spotting solution, in order of decreasing physical stability, are: Pronto! Universal Spotting Solution, 50% DMSO, 3 x SSC supplemented with 1.5 M betaine, 150 mM NaPO4, and 3 x SSC.

These solutions have successfully been used to fabricate DNA arrays on UltraGAPS slides. DNA dissolved in DMSO-containing media may aggregate if solvent concentration exceeds 70%, which happens upon prolonged use of the source plates and freeze-thaw cycling. Aqueous SSC- and NaPO4-containing media have a tendency to salt out, which may cause quill pins to clog, and require reconstitution after each print run, which leads to noticeable variability in DNA concentration among source wells.

Preparation of probe DNA.

Double-stranded DNA for spotting on microarrays is generally obtained by amplification of cloned coding sequences. It is important to purify the amplified fragments as the presence of primers and other components of the reaction mix may interfere with binding to the slide and produce background fluorescence upon hybridization.

Be sure to use purification methods that do not contribute fluorescent materials. Only oligonucleotides of the highest quality should be used for microarraying. The optimal length of oligonucleotides to be arrayed on UltraGAPS slides for transcriptional profiling is 70 nucleotides. As the GAPS-coated surface provides free amine groups for ionic attachment of the negatively charged phosphate groups of the DNA backbone, functionalization of the oligonucleotides with an amine or other reactive group is not necessary.

Oligonucleotides with and without such modifications bind equally efficiently to the UltraGAPS substrate.

Concentration of probe DNA.

The high reactivity of UltraGAPS slides allows the use of dilute printing solutions. The optimal concentration needs to be determined empirically. When too little DNA is used, the DNA spots will not reach signal saturation levels, thus reducing the dynamic range of the array. Conversely, highly concentrated printing solutions can produce spots with "comet tails" and other forms of localized background. The concentration and purity of the DNA should be checked spectrophotometrically as well as electrophoretically. The recommended concentrations to use as a starting point for further optimization are 0.15 mg/mL when spotting dsDNA (e.g., PCR products, genomic DNA) and 0.50 mg/mL when spotting oligonucleotides.

Immobilization procedures.

Binding of DNA to the GAPScoated surface is enhanced by UV cross-linking and/or baking. These procedures work equally well for DNA molecules longer than 300 bp. Smaller DNA molecules and oligonucleotides are best immobilized by UV cross-linking. When baking, care should be taken regarding the cleanliness of the oven. Volatile organics can irreversibly contaminate the surface of the array leading to high backgrounds.

Array Fabrication and Stabilization

1. Prepare source plates by dissolving purified probe DNA in the spotting solution. For double-stranded DNA, prepare solutions containing between 0.10 and 0.20 mg/mL. For oligonucleotides, prepare solutions containing between 0.35 and 0.70 mg/mL (the molarity of 0.5-mg/mL solution of unmodified 70-mers is 22 µM).

2. Set up arrayer and print slides according to manufacturer's or laboratory protocol. Always handle slides by the corners and wear powder-free gloves.

3. Remove arrays from printing platform and place them in original slide container or Corning 25 Slide Holder (Cat. No. 40081).

4. Incubate arrays in desiccator for 24 to 48 hours (vacuum desiccator works best).

5. (Optional see note) Rehydrate spotted DNA by holding slide (array side down) over a bath of hot purified water (95 to 100°C) for approximately 5 seconds until condensation of the water vapor is observed across the slide. Snap dry array by placing it (array side up) on a hot plate for 2 seconds.

6. Immobilize spotted DNA

For double-stranded DNA, use a UV cross-linker to apply 150 to 300 mJ of UV energy. Alternatively, bake the arrays at 80°C for 2 to 4 hours. If baking, place arrays in lidded glass container and make sure oven is clean and free of volatile organics. For oligonucleotides, use a UV cross-linker to apply 600 mJ of UV energy. Baking does not work well for oligonucleotide arrays.

7. Place arrays back in storage container and store in desiccator at ambient temperature. Arrays can be stored for up to 6 months prior to hybridization.

Exchanging the regular atmospheric air for clean nitrogen gas helps prevent oxidation of spotted material and extends the shelf life of the arrays.

Note: Rehydration and snap drying have historically been done to denature double-stranded DNA spotted in a non-denaturing medium and to evenly distribute the probe DNA within the spotted area. The efficacy of this treatment has not been conclusively demonstrated. If performing this step, utmost care must be taken not to overexpose the array to the hot plate, since doing so will affect the integrity of the spotted DNA and increase background. Do not place arrays in boiling water as this may result in a significant loss of probe DNA and delamination of the coating.

DNA precipitation (PCR concentration)

1. Transfer PCR reactions to 96-well V-bottom plates (POLY LABO ref.13456).

2. Add 1/10 vol. 3M sodium acetate (pH 5.2) + 1 volumes isopropanol. Store at -20°C few hours to overnight.

3. Centrifuge 3500 rpm for 1 hr

4. Remove supernatant with 12-channel pipetman

5. Add 100 uL of ice-cold 70% ethanol and centrifuge again for 30 min.

6. Dry the pellets in the centrifuge for 10 min.

7. Resuspend DNA in required volume of dH2O overnight.