Use RNAse free technique (skin and microorganisms are a source of RNAse) = always use gloves while touching or opening tubes, avoid talking or sneezing into tubes, bake all glassware, use DEPC treated H20 for all aqueous solutions, minimize exposure of pipette and tubes to air and dust, keep pipettors clean or ideally use filter tips. Keep RNA on ice at all times or store at -70C.
Cell Lysis
1. ____ Collect several backup samples for each experiment. Spin down cells
and immediately freeze, the best way is to quick freeze in liquid N2. Collect
15-20 ODU (Optical Density Units) for Exponential Phase cells and 30-40 ODU
for Stationary Phase and Diauxic Shift cells per sample. Store pellets at -70C.
Cell pellets from harvest date _________; ODU of cell pellets _________ .
2. To frozen pellet add:
____ 300ul of room temp. Cell Lysis Solution (Gentra)
____ Reporter gene RNA if desired, such as 2 ng of CAT mRNA, can monitor RNA
prep quality with a Northern or microarray spike
3. ___Transfer cells (frozen slush if possible) and solution to a tube used
for bead beating
Circle one => a screwcap tube or regular epi tube
4. ____ Add one scoopful of baked glass beads (0.5mm) using a homemade scoop
with a large PCR tube or two scoops of one lid from a PCR tube strip - about
0.5 ml.
5. ____ Use the bead beater in chromatography refridgerator (4ºC) (speed:
50X100 rpm) to lyse cells for 30 sec alternating with at least 30 sec on ice
(Do this 6 times).
cross number as you go =>: 1 2 3 4 5 6.
6. ____ Centrifuge for 3 min in refrigerated centrifuge. Transfer supernatant to prechilled, new eppendorf tube.
RNA precipitation:
1. ____ Add 100ul of room temp Protein-DNA Precipitation Solution (Gentra) to supernatant.
2. ____ Invert 10 times and put on ice for 5 min.
3. ____ Centrifuge 3 min (refrigerated centrifuge).
4. ____ Add supernatant to pre-chilled eppendorf tube containing 300ul of cold 100% isopropanol.
5. ____ Mix by inversion at least 50 times, mix well
6. ____ Centrifuge 3 min then remove supernatant. Note size and color of pellet.
7. ____ Resuspend pellet in 100ul of DEPC-H2O.
CH3Cl: Phenol Treatment To remove lipids, DNA and protein to improve UV quantitation
1. ____ Add 100ul of phenol:chloroform (5:1, water saturated, acidic with Isoamyl
alcohol -for example, pH 4.5 from Ambion) to RNA.
Please note brand and date of bottle __________________
2. ____ Vortex 1 min then centrifuge 2 min for a tight interface
3. ____ Remove up to 80ul of aqueous phase to fresh eppendorf tube avoid the interface and organic phase.
4. ____ Back-extract = Add 80ul of depc-H20 to phenol:chloroform and repeat steps 2 and 3. (See Ambion Tech Note: 5:4 pg.10 for additional information)
5. ____ Combine aqueous phases (i.e. RNA) from steps 3 and 4.
6. ____ EtOH precipitate the RNA by adding:
" 0.1 times volume of 5M NH4OAc = _____ul
" 2.5 times volume 100% EtOH = _____ul
7. _____Precipitate overnight at -20oC. This is not crucial, but definitely
helps get everything to precipitate. 1-2 hours at -20oC, and in a crunch, 15-30
min at -70oC works great too.
Precipitation time __________.
8. ____ Centrifuge precipitated RNA 20 min in refrigerated centrifuge. Note orientation of tube to keep track of pellet.
8. ____ Remove supernatant and wash RNA pellet with 0.5 ml of 70% RNase free EtOH. Vortex 1 min or until pellet is loosened from bottom.
9. ____Centrifuge 10min and remove EtOH.
10. ____ Centrifuge additional 1min and remove residual EtOH.
11. ____Dry pellet 10-15min in air (place a kim wipe in a tip box, then open epindorf tube caps and turn the tubes upside down on the kim wipe).
12. ____ resuspend in 50ul DEPC-water. Store at -70oC
Storage location ___________________________.
Quantitation (Pre-Rneasy Quantitation)
Quantitate all RNAs with the spectrophotometer. Dilute RNA in 10mM Tris-HCl pH 7.5 to so that peak absorbance at 260 nm is <1.2 OD. (typically 1:50, dilution factor (DF)= 50). Use the 100 l cuvette. DF=__________
Calculate Concentration of stock and Total yield:
[RNA]ug/ml x 1ml = (value) x DF = Concentration of stock (ug/ul)
1000ul
Then take: Concentration of stock (ug/ul) x Total volume resuspended RNA in = Total yield(ug per ul(vol. resuspended)).
Sample name Nucleic Acid (ug/mL) Dilution Factor
(DF) Concentration of stock Total volume Total yield
Determine if there is Polysaccharide Contamination:
A260 =(value) Note: If (value) is below 2 the RNA may have
A230 polysaccharide contamination.
Sample name A260 A230 A260/A230 Polysaccharide contamination (Yes/No)
Rneasy (Qiagen) Cleanup To remove residual phenol (Rneasy Mini protocol for
RNA cleanup: steps 1-3 (4/2000 handbook))
The Rneasy column can hold a maximum of 100 ug.
1. ____ Adjust sample to a volume of 100 µl with RNase-free water. Add 350 µl Buffer RLT (toxic -me is not necessary, if you use it, do in the hood and dispose of properly), and mix thoroughly. Volume of H20 added ____________.
2. ____Add 250 µl ethanol (96-100%) to the diluted RNA, and mix thoroughly
by pipetting.
Do not centrifuge. Continue immediately with step 3.
3. ____Apply the sample (700 µl) to an RNeasy mini column placed in a
2 ml collection tube
(supplied). Close the tube gently, and centrifuge for 15 sec at 8000 x g ?<10,000
rpm (usually do 13,000 rpm). Discard the flow-through and collection tube.
DNaseI Treatment (steps 1-8 correspond to steps D1-D8 from the Rnase-Free-DNase Set (Qiagen) Protocol (1/1999) Prepare DnaseI stock according to the manufacturer's protocol
1. ____ This step is not necessary if protocol was done as written above. This step is suggested when starting at this point with whole cell lysate or RNA that has not gone through such a rigorous cleaning. Pipet 350 ul Buffer RW1 into the spin column, and centrifuge for 15 sec at _8000 x g to wash. Discard flow -through and reuse collection tube.
2. ____ Add 10 l DNase I stock solution to 70 l Buffer RDD. Mix by gently inverting the tube (If working with several samples, prepare a master mix and add 80ul).
2. ____ Pipet the DNase I incubation mix (80 l) directly onto the spin-column membrane and place on the benchtop for 15 minutes.
3.____ Pipet 350 l Buffer RW1 into the spin column and centrifuge for 15 seconds at < 8000 rpm (usually 13,000 rpm). Discard flow-through and collection tube.
4. ____ Place the spin column in a new 2-ml collection tube. Pipet 500 l of Buffer RPE into the spin column and centrifuge for 15 seconds at < 8000 rpm (usually 13,000 rpm). Discard flow-through. Reuse collection tube.
5. ____ Pipet 500 l Buffer RPE into the spin column and centrifuge for 2 minutes
at maximum speed. Discard flow-through. Reuse collection tube.
6. ____ Spin 30 seconds to dry the silica-gel membrane. Remove column from collection
tube. Make sure the column does not touch the ethanol flow-through!
7. ____ Transfer the column to a new 1.5 ml collection tube and pipet 30ul of depc-H20 directly onto the silica-gel membrane. Centrifuge for 1 minute at < 8000 rpm (usually 13,000 rpm).
8. ____ Repeat step 7 if the expected RNA yield is >30 g (this 2nd elution
is typically performed).
Total Volume _____________.
Quantitation (Post-Rneasy Quantitation)
Quantitate all RNAs with the spectrophotometer. Dilute RNA in 10mM Tris-HCl pH 7.5 to so that peak absorbance at 260 nm is <1.2 OD. (typically 1:50, dilution factor (DF) = 50). Use the 100 l cuvette. DF=__________
Calculate Concentration of stock and Total yield:
[RNA]ug/ml x 1ml = (value) x DF = Concentration of stock (ug/ul)
1000ul
Then take: Concentration of stock (ug/ul) x Total volume resuspended RNA in = Total yield(ug per ul(vol. resuspended)).
Sample name Nucleic Acid (ug/mL) Dilution Factor
(DF) Concentration of stock Total volume Total yield
Determine if there is Polysaccharide Contamination:
A260 =(value) Note: If (value) is below 2 the RNA may have
A230 polysaccharide contamination.
Sample name A260 A230 A260/A230 Polysaccharide contamination (Yes/No)
Store at -70C Storage location _____________________________________________.
NOTES:
Lysing the cells and yield expected
Collect several backup samples for each experiment. Collect 15-20 ODU (Optical Density Units) for Exponential Phase cells and 30-40 ODU for Stationary Phase and Diauxic Shift cells per sample. Store pellets at -70oC. With bead-beating, 28-40 ODU stationary phase cells yield about 150- 300 ug of total RNA. For some experiments, washing the pellet is not feasible if one is doing a very tight time course. Angelina and Juanita elected to immediately freeze the unwashed pellet in liquid nitrogen after pouring off the supernatant (media).
Cell lysis by glass beads and minibeadbeater (Biospec - www.biospec.com) Screw top tubes prevent leakage, but 1.6ml microfuge tubes allow better visibility. If you choose the 1.6ml flip top tubes, you must clear the lip of all beads and parafilm the top to prevent leakage. Bead beater must be modified with the adapter screw for the 1.6 ml tubes.
Phenol Extraction
Add 100ul of CH3Cl: Phenol (saturated 5:1; pH 4.5-Ambion) to RNA. Isoamyl alcohol
encourages a tight interface, most phenol mixes contain it.
Remove 80ul of aqueous phase to fresh eppendorf tube. Do not attempt to remove all of the aqueous phase sample because undesirable products from the organic phase may also be drawn up. Back extraction is often suggested. This entails adding 80ul more water after removing the 80ul, vortexing, then removing 80ul.
CHCl3 : Phenol treatment to remove lipids, DNA and protein to improve UV quantitation. Pure RNA is critical for efficient cDNA synthesis. Although, DNA contamination should not be a problem during first strand synthesis its presence could yield inaccuracies during RNA quantitation and will reduce microarray slide hybridization. Qiagen Rneasy column to remove residual phenol
About Qiagen kits:
Rneasy Mini protocol for RNA cleanup: steps 1-3 (4/2000 handbook) There is no need to add Beta-mercaptoethanol, since, at this point, we are not using cell lysate but rather a pretty clean RNA prep. The lack of Beta-mercaptoethanol (B-me), an Rnase inhibitor, has been tested and shown to not affect the quality of the RNA, or reduce the quantity any less than 10%, (In the range of recovery variation) as seen by two experiments. If you choose to use -me, do your work in the hood, and dispose of your -me supernatants in an appropriate waste container.
Rnase-Free-DNase Set Protocol: steps D2-D8 (1/1999 protocol), This does not include steps 4-5 and step D1 respectively since we are using these kits for RNA that is already pretty clean. We have found that g the same clean RNA in DEPC treated water versus in Nuclease Free water (Rneasy kit) gives a 260/280 ratio of 1.8 and 2.0 respectively. We think this is related to the pH of the water, as described in the Rneasy protocol book.
Storage of RNA
For long term storage in 50ul of RNA Storage Solution (Na Citrate, pH 6.4; Ambion), however it is not known how this affects labeling. If RNA is stored in Na Citrate, it must be ethanol precipitated and resuspended in water. Note : a typical storage solution containing formamide
HYBRIDIZATION
Hybridization of labeled target onto Yeast 70-mer oligos printed on GAPSI and
UltraGAPS using Dextran sulfate and mixing.
Rev. 9/21/03
Monica Manginell, Anthony Aragon, Juanita Martinez
Margaret Werner-Washburne, Department of Biology, University of New Mexico.
NOTE! This document was created using Word 9 on Windows 2000. If you open this on a mac, the symbol for mu (micro) might turn into a dash, or something else. Keep an eye out for this.
REAGENTS
Oligo dT primer 1 mg/ml (Research Genetics)
FluoroLink Cy3-dCTP and Cy5-dCTP (Amersham)
Superscript II (Life Technologies)
Rnase A (USB)
Rnase H (Invitrogen)
dNTPs
Salmon Sperm DNA (1 ml stock from Gibco BRL 10 mg/ml)
DEPC treated water (made in lab)
LifterSlips (Erie Scientific) different sized available, we use 22x30 for 30
ul
50 ml Falcon tubes (VWR)
Formamide (Deionized, Omnipur EM science)
BSA fraction V(Sigma), 10 mg/ml stock made in lab and frozen
SDS (10% stock)
SSC (20 x stock pH 7.0, filter sterilized, made in lab)
0.2 m Nylon filters for filter sterilization
115V Nutator Mixer (Clay Adams)
LABELING
We typically synthesize Cy3, Cy5 labeled cDNA from 20 µg of total RNA
from stationary phase or log phase yeast S. cerevisiae using direct Cy-dCTP
incorporation by Superscript II reverse transcriptase
We follow the section "cDNA synthesis" in the protocol "Detailed
Instructions for Hybridization of cDNA Probes to dsDNA Arrays made on GAPS II
Slides" rev. Feb 2002 (Corning),below with the following exceptions:
To your RNA/primer annealing mix, add spike mRNA (some print runs contain arabidopsis genes spots, you may need to determine the amount of spike you will use)
We decided that using the tetrad thermocycler with the hot-lid option (in the arrayer room) will give optimal and consistent results. Please indicate your PCR machine and method.
Labeled target is typically stored in pH8 water at 4ºC for up to 2 weeks (no DTT), although target as old as one month was found mostly intact.
We clean our microcuvette with ddH20 or blank solution, and occasionally with 1% Helmanex and an aspirator setup. It is best to not use the sample that you put into the cuvette for hybridization.
NOTE: RNase treatment: 0.5 µl of RNase A (USB) is still used even though the concentration of the stock we buy is about 10 fold more than 3.2 U/µl as indicated in Cornings Protocol. Corning acknowledges this is confusing, however the actual amount of Rnase to digest 20 µg of total RNA needs to be investigated. Juanita has reason to believe that not all the RNA gets degraded. This may cause quantitation error for labeled target, but as far as we know, it is not affecting the hybridization.
cDNA-Synthesis Protocol
A. Synthesis of cyanine-labeled cDNA
1. Program a thermocycler as follows (waterbaths may be used as an alternative to a thermocycler):
Temperature Time Reaction
70 C 10 min Denaturation of template RNA
4 C 5 min (at this point, add master mix, pause if necessary)
23 C 10 min Annealing of primer to RNA
42 C 2 h Extension of cDNA chain
4 C hold
2. Depending on the RNA template, prepare the appropriate RNA/primer annealing mix on ice:
For Total RNA Templates
Total RNA 20 µg
Oligo(dT) primer 2 µg
Nuclease-free water to 23 µl
For mRNA Templates
mRNA 1.5 µg
Random primers (9-mers) 3.3 µg
Nuclease-free water to 23 µl
3. Place tubes in the thermocycler and initiate the cycling program.
4. Perform this step and step A5 during incubation of the RNA/primer mix. Prepare
working stock of dNTPs having the following concentrations: 0.5 mM dATP, dGTP,
dTTP, and 0.25 mM dCTP.
5. Keeping all reagents on ice, add in the following order (if performing multiple
cDNA synthesis reactions, a single labeling mix can be prepared by scaling up
the volumes proportionally):
Cy3™ labeling Cy5™ labeling
5 X first-strand buffer 8 µl 8 µl
0.1 M DTT 4 µl 4 µl
dNTPs (from working stock prepared in step A4) 2 µl 2 µl
1 mM FluoroLink™ Cy3™-dCTP 1 µl --
1 mM FluoroLink™ Cy5™-dCTP -- 1 µl
SuperScript™ II Reverse Transcriptase (200 U/µl) 2 µl 2 µl
17 µl 17 µl
6. Initiate the cDNA synthesis reaction by adding the labeling mix from step A5 to the RNA/primer mix of step A3, during the initial 5 minute hold at 4 C. The final reaction volume of the reaction is 40 µl. Continue the thermocyler program (10 min at 23ºC followed by 2 h at 42ºC).
Note: The composition of the labeling reaction has been optimized to yield sufficient cDNA for probing up to 2 full-size arrays using a minimal amount of Cy-dCTP. Reactions carried out as described usually yield 200 ng of cDNA containing 15 to 20 pmoles of incorporated Cy-dCTP. The same amount of template RNA typically yields 400 to 550 ng of cDNA of similar fluorescence strength when the concentration of the dNTP stock is raised to 5 mM dATP, dGTP, dTTP, 2.5 mM dCTP, and 5 ul of Cy-dCTP is used.
B. Removal of the RNA Template
The RNA template must be eliminated by digestion with ribonucleases to yield
single-stranded DNA (use a thermocycler or water baths).
To the labeling reaction from step A6 add 1 µl of RNase H and 0.5 µl of RNase A.
Incubate at 37 C for 15 min.
Note: Treatment of the mRNA/cDNA hybrids with NaOH also eliminates the ribonucleic acids. However, RNase digestion is recommended over NaOH treatment because it produces higher yields of labeled cDNA.
C. Probe Purification
1. To precipitate the labeled cDNA, add 4 µl of 3 M sodium acetate, pH
5.2, to the 41.5-µl cDNA-synthesis reaction from step B2, mix briefly,
then add 100 µl of 100% ethanol. Briefly mix the contents of the tube
and incubate at -20ºC for at least 30 min. The cDNA may be stored at -20ºC
for up to one week. This is a good stopping point if the purification procedure
cannot be completed within the day. Following incubation at -20ºC, centrifuge
at 12,000 x g for at least 30 min to pellet the cDNA.
2. Carefully remove the supernatant and dissolve the pellet in 40 µl of
nuclease-free water. Add 4 µl of 3 M sodium acetate, pH 5.2, and 200 µl
of QIAquick™ Buffer PB. Incubate at 37ºC for 15 min (incubation at
95ºC for 30 sec is sometimes necessary to fully dissolve the pelleted cDNA).
3. Place a QIAquick™ column in a 2-ml collection tube. If combining several
labeling reactions, keep in mind that these columns have a binding capacity
of approximately 10 µg. For the purpose of purification, assume a yield
of 1 µg of cDNA per reaction. Apply the cDNA/Buffer PB mixture to the
center of the column.
4. Centrifuge column at 14,000 rpm for 60 sec in a microcentrifuge.
5. Discard flow-through and place column back in the collection tube.
6. Add 600 µl of QIAquick™ Buffer PE to the column. Centrifuge at
14,000 rpm for 1 min or until buffer has passed through column.
7. Discard flow-through and place column back in the collection tube.
8. Repeat steps C6 and C7 once more for a total of two 600-µl washes with
Buffer PE.
9. Centrifuge the column for 2 min at 14,000 rpm to dry off the ethanol.
10. Place the column in a clean 1.5-ml microfuge tube. Apply 35 µl of
elution buffer (we recommend the use of distilled water with pH adjusted to
8.0 by addition of a small volume of NaOH) preheated to 37ºC to the center
of the resin bed and let stand for 1 min.
11. Centrifuge column at 14,000 rpm for 1 min.
12. Repeat the elution step with a second 35 µl of pH8 water.
Note: The QIAquick™ PCR columns eliminate single-stranded DNA molecule less than 200 nucleotides long (100 bp for double-stranded DNA). Failure to remove these small fragments from the probe may result in higher backgrounds.
D. Determination of the Frequency of Incorporation of Fluorescent Nucleotide into the labeled target.
We typically spec 10 l of the cleaned labeled target in the quartz microcuvette. This sample is not added back to the sample for hybridization so as to not risk contamination, but can be used for other test hybridizations. Please see Corning protocol (Detailed Instructions for Hybridization of cDNA Probes to dsDNA Arrays made on GAPS II Slides" rev. Feb 2002 section D, page 3). This should be used as a guideline to determine whether labeling was successful or not, but not as a quantitative measure of labeling. Angelina and Juanita observed residual free Cy-dCTP carryover after cleanup and this affects the spectrophotomer scan. It is a good idea to compare your labeled samples with a no-RT control.
HYBRIDIZATION
1. Pre-scan all of your chosen slides at 600 PMT and 10 µm resolution (normal scan) and save files. Visually check for the presence of background and make a decision about the quality, depending on your own standards. 50-80% of the green background speckles will be removed in the prehyb. Use the scan and GenePix to ascertain the distance (10,000) µm = 1 cm) of the print blocks from the top of the slide, which changes for every print run. The cover slip should be positioned accordingly.
2. Prepare solutions.
The following solutions may form precipitates if chilled, which may fluoresce
and ruin your data. Since a small volume of the hyb solution is actually used
per slide (about 30ul), making a small amount, even 1ml, fresh every time may
introduce day to day variation due to pipetting if one is not VERY careful,
also we don't want to make too much because it will go to waste. Therefore,
follow these guidelines
Prehybridization solution
80ml per coplin jar Please note changes here
50% Formamide 40 ml stock
5 x SSC 20 ml of 20X stock
0.1% SDS 800 µl of 10%
0.1 mg/ml BSA 800 µl of 10 mg/ml
ddH20 18.4 ml ddH20
Add Formamide, water, SDS and SSC, filter sterilize with 0.2 µm Nylon filter, then add BSA
Hybridization solution
Make 10 ml Premix Please note changes here
50% Formamide 5 ml stock
5% Dextran sulfate 0.5 g
5X SSC 2.5 ml (20X stock)
0.1% SDS 100 µl (10% SDS stock)
0.1 mg/ml BSA 100 µl (10 mg/ml stock)
ddH20 2.2 ml ddH20
100 µg/ml Salmon sperm DNA -----
Add Dextran sulfate to water and dissolve at 37-42ºC. Add formamide, SSC,
and SDS. Filter sterilize with 0.2 µm Nylon filter (can use luer-lock
syringe with a filter), then add BSA
Hybridization solution (enough for 9 slides)
297 µl premix
3 µl Salmon Sperm DNA (10 mg/ml stock)
3. Set prehybridization solution in a coplin jar in the 42ºC water bath
and let sit at least ½ hour before proceeding to the next step.
4. The following three steps should be done concurrently
A. Prehybridize slide in 50 ml of Prehybridization solution in a Coplin Jar at 42 C for 45min-1hour. (prehybridization for 1.5 hours has been done and is suspected to remove a considerable amount of background) Rinse the slide briefly (5 sec) with ddH20 from a squirt bottle and then dip in 100% ethanol for 1 second. Dry under 15-30psi N2 gas stream to push off all droplets of liquid. Do not allow liquid to dry on the slide. Slides are used immediately for hybridization. Do not let prehybed slides sit dry for more than about ½ hour before hybing.
B. Clean the cover slips while the slides are undergoing prehybridization. Dip and swirl each coverslip (about 5 secs) in a 50% ethanol, 1M KOH solution (in a coplin jar), then rinse coverslips briefly with ddH20 wash bottle then dry with N2 gas. The goal is to have a nice sheeting action and no streaks. Keep in dust free box until use).
C. Turn the heat block on to 95ºC.
Keep labeled target in the dark whenever possible. We typically turn out the lights whenever possible. We use aluminum foil to wrap tubes, and racks and cover the lyophilizer. However, it is often not convenient to cover while adding labeled target to the slide or during short washing steps.
5. Prepare labeled target for the hybridization
For data collection, adding the entire preparation of cDNA from the 20 µg
of RNA to the slide for hybridization is the best approach, simply because 1)
sometimes we cannot confidently quantitate the number of pmols of labeled target
we are adding and 2) altering the amount of cDNA produced in a RT reaction may
alter results. We have the opportunity later to equilibrate the signals of the
two dyes by adjusting the PMT settings during scanning.
Dry labeled target in speed vac (lyophilizer) with heat for about 1 hour, or
until dry. Do not over dry. Resuspend in the volume of hybridization buffer
appropriate for the size of your coverslip.
Heat mixture to 95 C for 5 minutes to denature DNA, then spin for 30 sec.
Our suggested volumes for coverslips (practice first!):
22X30I LifterSlip 30 l amount used_______ ( 4 pin format)
24X30I LifterSlip 30-35 l amount used_______ (16 pin format)
6. Begin hybridization - work without haste or interruption
Dextran sulfate is very viscous. Various methods can be used to apply the probe
to the slide.
please indicate yours:
1) pipet labeled target onto the underside of the coverslip which is set on a flat clean surface (the back of the chamber works) (avoid dispensing bubbles) then drop the coverslip onto the slide (see Juanita). If bubbles are formed use a dry coverslip or tip to pop them.
2) apply the probe to the slide in two or three drops over the array. Take great care to avoid dispensing bubbles. If bubbles are dispensed, carefully pop them with the corner of a dry coverslip, avoid getting too much liquid on the coverslip and DO NOT touch the array. Place the cover slip on top taking care that the coverslip is aligned properly (see Monica).
Another way to avoid bubbles is to dispense just to the end of the tip, and not blow out. This must be consistent to avoid volume variation. (1µl loss from 30 = 3%)
In all methods the target solution will disperse via capillary movement. Experiment and practice FIRST to determine how you will be most successful. When you practice be sure to use a hybridization /dextran sulfate solution.
Enclose the slide in a CMT chamber. Add 10- 12 l _______ of water to the hydration wells. Tape the closed chambers to the orbital platform (nutator) in an oven set to 42 C and incubate for 4 to 20 hours. Time start _______ ; Time begin wash__________; Total time hybed __________
Using Dextran sulfate and mixing, we saw adequate signal after 4 hours, but did not linearly increase at 20 hours. Overnight is suggested, but 4 hours is the minimum for detectability. Complete hybridization is the goal.
WASHING
# of slides washed at once_________
Based on "Detailed Instructions for Hybridization of cDNA Probes to dsDNA Arrays Made on GAPS II Slides" rev. Feb 2002, Corning. Wash buffers should be filter sterilized when possible to remove dust. Prewarm 2X SSC wash buffers to 42 C before beginning. Reduce light exposure to slides whenever possible. It is difficult to wash more than 4 slides at once, if so, these are minimum times.
We use one 50 ml Falcon tube per slide per wash step. Fill falcon tube to 1 cm from the top. As the slide is added to the tube, keep track of the orientation.
The first wash is typically left in 42ºC water bath without shaking. Cover slip should come off during this wash, if not, gently rock by hand. Do not allow the coverslip to scratch the slide surface.
For slides "on rocker", place each falcon tube on an orbital platform (nutator) with the length of the tube parallel to the length of the rocker and with array side up, and cover with foil to keep light out and heat in, if any.
After the last wash, dry under 15-30 psi stream of N2 gas. Place back in clean chamber or slide holder and scan.
Wash Buffer (Temp.) Wash Time How Number of Washes
2 x SSC, 0.1% SDS (42ºC) 1 min in 42ºC bath 1
2 x SSC, 0.1% SDS (42ºC) 5 min on rocker 1
0.1 x SSC, 0.1% SDS (RT) 10 min on rocker 1
0.1 x SSC (RT) 10-20 sec dip or invert 1
0.1 x SSC (RT) 2 min on rocker 2
0.1 x SSC (RT) 1 min on rocker 1
0.01 x SSC (RT) 15 sec dip or invert 1