Large-scale PCR for cDNA microarrays
Large-scale PCR for cDNA microarrays
This protocol describes the experimental procedure to make cDNA microarrays by PCR amplification of products. This experiment was derived from PCR Lab Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Large-scale PCR for cDNA microarrays
Materials and Instruments
Buffers, solutions and reagents Biomolecules Enzymes and enzyme buffers Nucleic acids and oligonucleotides Gels Move Phase 1: Preparation of Photocopy Layout and Work Bank For more product details, please visit Aladdin Scientific website.
Specialty equipment
I. Materials
1. Buffers, solutions and reagents
Carbenicillin (100ug/ml storage solution)
Ethanol (100%)
Glycerol, 45% (v/v)
LB liquid medium. (1L of LB liquid medium contains 10 g tryptone, 5 g yeast extract, 5 g NaCl)
Super Liquid Medium. (1L Super Liquid Medium contains 32 g tryptone, 20 g yeast extract, 5 g NaCl, 5 ml 1 mol/L NaOH)
2. Biomolecules
Parental clones or ESTs with checked sequences (e.g., gf211release, ResearchGenetics)
3. other instruments
Centrifuge with horizontal (bucket) head, 6.2 cm deep, capable of centrifuging microtitration and filtration plates (e.g., Sorvall SuperT 21, Sorvall)
Airpore Tape Sheets (Qiagen)
Bunsen Torch
Incubator, preset to 37°C
96 Needle Inoculator (V&P Scientific)
Deep 96-well microtitre plate, 1.0 ml,PP (V WR International)
U-Bottom 96-well Microtitration Plate (Corning)
Tissue paper
Oscillatory incubator with deep-well plate holder, 37°C
Zip-lock bag, 1 gallon (~4L)
II. Methods
1. Incubate sealed maternal clone plates at 37°C overnight.
2. Prepare working replicates for clonal line preparation: label 96-well round-bottomed (U-bottomed) microtitre plates and dispense 100ul of LB Liquid Medium containing 100ug/ml Carbenicillin into each well.
Maternal clones were maintained with these working copies. Tests were performed to ensure that these clones all had vector-conferred ampicillin resistance.
3. Centrifuge the plate of maternal clones in a horizontal microtitre plate rotor at 1000 r/min for 2 min to remove condensation from the plate seals.
4. Fill a small beaker with some 100% ethanol and soak the 96-pin replication tool in ethanol. Then remove the tool from the ethanol and burn the inoculated needles.
5. After cooling briefly, dip the replication tool in a maternal cloning plate and transfer to a working cloning plate. If necessary, repeat the inoculation once for each plate.
6. Cover the inoculated LB plates and place them in a 1-gallon (3.78541 dm3 ) zip-top bag with wet paper towels, seal the bag and incubate overnight at 37C.
7. Dispense 1ml of Super Liquid Medium containing 100ug/ml Carbenicillin into each well of the deep well plate.
These plates will be used as a source of culture medium for the preparation of templates.
8. After 1 day, inoculate directly from fresh LB plates to deep well plates using the replication kit.
9. Seal the deep-well plates with Qiagen Airpore Tape Sheets, cover with a plastic lid and incubate for 24 h at 200r/min in a 37°C oscillating incubator.
10. For plates that are to be frozen (-80°C) as a source of subsequent cultures, add 5ul of 45% (v/v) sterile glycerol to each well. Isolate the plasmid template from the remaining plates.
Phase 2: Isolation of plasmid templates
I. Materials
1. buffers, solutions and reagents
Ethanol, 70 percent
T low E buffer (10 mmol/LTris-HCl, O.lmmol/LEDTA, pH 8.0)
2. centrifuge and rotor head
Centrifuge with horizontal (bucket) rotating head, 6.2 cm deep, for centrifugation of microtitre plates and filter plates (e.g. Sorvall SuperT 21, Sorvall).
3. Specialized equipment
Plate sealer
Vortex mixer
4. Carriers and bacterial strains
Bacterial culture obtained from step 9 of phase 1 above
& additional reagents
AlkalineLysisMiniprepkit,96 wells (EdgeBiosystems)
Methods
1. Heat the Lysis Buffer (Edge Biosystems Kit) to 37°C to dissolve the SDS.
2. Add lml of RNAase solution to 100 ml of Resuspension Buffer (Edge Biosystems Kit). Store at 4°C for up to 3 months.
3. Add 350ul of Bacterial Culture Solution to each well of the EdgeBiosystemsKit Receiver Plate. Place the filter plate on top of it and secure it with a band.
4. Centrifuge the bacterial culture in the deep-well plate at 1500 g for 7 min using a centrifuge fitted with a 96-well plate rotary head.
5. quickly invert the plate on a clean paper towel and gently snap out the remaining culture medium.
Do not delay this step or the precipitate will come loose and may be lost when pouring out the medium.
6. Resuspend each precipitate with 100ul of Suspension Buffer and vortex to mix until all precipitates are suspended.
Inadequate suspension will result in lumpy cells that will not lyse in subsequent steps.
7. Add 100ul of Lysis Buffer to each well and mix by gently shaking the plate for lmin to avoid breaking the bacterial chromosomal DNA.
8. Add 100ul Precipitation Buffer to each well and mix for 1 min.
9. Add 100ul Neutralization Buffer to each well and vortex for 20s.
10. Transfer the mixture from the deep-well plate to a pre-assembled filtration/reception assembly plate.
11. Centrifuge the stacked plates at 1500 g for 12 min in a centrifuge equipped with a 96-well plate rotary head.
12. Remove and discard the filtration plate, pour the ethanol and filtrate from the receiving plate, and blot the remaining ethanol on a clean paper towel.
13. Add 500ul of 70% ethanol to each well, pour out immediately and blot the remaining ethanol on a clean paper towel.
14. Remove the lid and place the plate in a clean drawer and cover with a clean paper towel and dry overnight.
15. Suspend each DNA precipitate by adding 200ul each of T low E buffer, seal the top of the plate with a plate sealer and allow the precipitates to rehydrate at 4°C for at least 2 days. For long-term storage, keep at -20°C.
Phase 3: Amplification of clones and evaluation of PCR products
I. Materials
1. Buffers, solutions and reagents
DEPC-treated H20
TAE buffer, 1× (40 mmol/L Tris-acetate, 1 mmol/L EDTA, pH ~8.5)
DNA Spotting Buffer (200 g/L Ficoll400, 0.1 mol/L Na2EDTA, pH 8.0, 10 g/L SDS, 2.5 g/L Bromophenol Blue, 2.5 g/L Xylene Nitrile)
dATP solution, 100 mmol/L (Pharmacia)
dGTP solution, 100 mmol/L (Pharmacia)
dTTP solution, 100 mmol/L (Pharmacia)
dCTP solution, 100 mmol/L (Pharmacia)
2. Enzyme and enzyme buffer
AmpliTaq DNA polymerase (Perkin-Elmer)
GeneAmp PCR buffer, 10X (Applied Biosystems)
3. Nucleic acids and oligonucleotides
Carrier or gene-specific primers (1 mmol/L)
100bp DNA ladder (New England Biolabs)
4. gel
Gel (20 g/L, TAE buffer)
5. Specialized equipment
Thin-walled PCR plates and Cycleseal PCR plate sealer (Robbins Scientific)
Electrophoresis unit for 4 x 50-well combs
Thermal cycler
6. Additional equipment
Equipment and reagents for agarose gel electrophoresis, including ethidium bromide
1. Prepare a master PCR mix for each 96-well plate to be amplified containing the following components.
PCR buffer, 10X (containing 15 mmol/LMgCl2 ) 1000ul
dATP (100 mmol/L) 20ul
dGTP (100 mmol/L) 20ul
dCTP(100mmol/L)20ul
dTTP(100mmol/L)20ul
Primer 1 (lmmol/L) 5ul
Primer 2(lmmol/L)5ul
AmpliTaq Polymerase (5U/ul) 100ul
DEPC-treated H20 8800ul
2. Label the 96-well PCR plate and dispense 100ul of PCR mix into each well.
Gently snap the PCR plate to ensure that no air bubbles remain at the bottom of the wells.
3. Add 1ul of purified plasmid template to each well and close the lid.
4. Perform the following cycle. 
Store the plate at 41°C while performing the gel analysis for the mass control.
5. Detect PCR products by agarose gel electrophoresis.
Gel imaging allows only crude quantification of the product, but provides a good characterization of the product composition.
6. 2ul of each well is mixed with 2ul of Spotting Buffer.
7. Analyze samples on a 2% agarose gel using a 100bp DNA ladder as a standard.
Each lane should show a single major band and the brightness of the product should be the same from lane to lane.
Stage 4: Purification and setting of PCR products
I. Materials
1. Buffers, solutions and reagents
Ethanol/acetate mixture (95% ethanol, 5% 3 mol/L sodium acetate, pH 6.0)
Ethanol, 70
SSC buffer, 20X (3 mol/L NaCl, 0.1 mol/L sodium citrate-2 H20 with 1 mol/L HCl, adjust pH to 7.0)
TAE buffer, 1X (40 mmol/L Tris-acetate, 1 mmol/L LEDTA, pH about 8.5)
DNA Spotting Buffer (20% Ficoll400, 0.1 mol/L Na2EDTA, pH 8.0, 10 g/L SDS, 2.5 g/L Bromophenol Blue, 2.5 g/L Xylene Cyanide)
2. Centrifuge and rotor head
Centrifuge with horizontal (bucket-type) rotating head, 6.2 cm deep, capable of centrifuging microtitration and filtration plates (e.g., SorvallSuperT21, Sorvall).
3. Nucleic acids and oligonucleotides
100bpDNAladder (New England Biolabs)
Double-stranded DNA standard with variations of 0, 50ug/ml, 100ug/ml, 250ug/ml, 500ug/ml
Reference double-stranded DNA (0.5ug/ml) (Invitrogen)
4. gels
Agarose gel (20 g/L, TAE buffer)
5. Specialized equipment
V-bottom 96-well microtitration plate (Corning)
Immunowash microtitration plate washer (BioRad)
Paper towels
Heat-sealable storage bags and heat sealers
65°C Incubator
Electrophoresis unit for 4 x 50-well combs
Electrophoresis power supply
96-well plate for fluorescence detection (Dynex)
Fluorometer (e.g., Perkin-Elmer Analytical Instruments)
6. Additional equipment
FluoReporter Blue dsDNA Quantitation Kit (Molecular Probes)
II. Methods
1. Add 200ul of ethanol/acetate mixture to each well of a V-bottom 96-well plate.
2. 100ul of PCR product from each well was transferred to a V-bottom plate and mixed by pipetting four times with a 75ul pipette.
3. Place the plate in the refrigerator at -80°C for 1h, or store at -20°C overnight.
4. Thaw the plates to reduce their brittleness and to dissolve any ice that may have formed in the wells.
5. Place the plate in a centrifuge with a microtitre plate rotor and centrifuge at 2600 g at 4°C for 40 min.
6. Aspirate the supernatant from each well with the Immunowash Microtitre plate washer.
It is recommended to leave approximately 10-20 ul at the bottom of each well to avoid stirring up the sediment.
7. Add 200ul of 70% ethanol to each well using the Immunowash Microtitre plate washer.
8. Centrifuge at 2600 g for 40 min at 4°C.
9. Aspirate the supernatant from each well using an Immunowash Microtitre plate washer.
10. Cover the plate with paper towels and dry overnight in a closed drawer.
1. Resuspension of PCR products
11. To each well, add 40ul of 3XSSC. Seal the plate with a sheet sealer or suitable sealer, taking care to seal each well tightly.
The buffer used to resuspend the PCR product is determined by the spotter and chip used to make the microarray.
12. Place the plate in a heat-sealed bag containing 3 X SSC moistened paper towels.
13. Place the bags in an incubator at 65°C for 2 h. Turn off the incubator and allow the plates to cool gradually.
Cooling the plates in the incubator prevents condensation on the seals.
14. Take 1ul of resuspended PCR product and analyze on a 2% agarose gel as described in stage 3.
Sufficient precipitation and resuspension will produce very bright bands.
15. Plates containing the resuspended PCR product are stored at -20°C.
2. Quantification of PCR products by fluorimetric assays
16. 200ul of Fluorophore Buffer (FluoReporter Blue dsDNA Quantitation Kit) is added to each well of a 96-well plate designed for fluorometric assays.
17. Add 1ul of PCR product to each well of the fluorescence assay plate.
18. Add 1ul of double-stranded DNA standard (0, 50ug/ml, 100ug/ml, 250ug/ml, 500ug/ml dsDNA) to the last row of the plate.
19. Set the excitation light of the fluorometer to 346nm and the emission light to 460nm.
20. Test the standard for linearity and reproducibility in the range of 0 to 500ug/ml dsDNA.
21. Subtract the value for the 0ug/ml sample from the values measured for all controls and other samples and follow the equation below.
The concentration of dsDNA in the PCR product is calculated using the following equation. 
22. Based on the above calculations, adjust the concentration of the PCR product if it is outside the range of expected printing concentrations (0.1-0.5ug/ul).
Stage 5: Printed microarrays
Upon completion of the previous protocol, the researcher will be provided with the necessary spotting material to produce the microarrays. The protocol parameters for printing microarrays are determined by the dispenser and chip used. Since there are several combinations of dispensers and microarrays that can be used to produce microarrays, detailed protocols for the dispensing of PCR products are not given here, but can be found elsewhere (Bowtell and Sambrook 2003).