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MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
PCR AMPLIFICATION OF YEAST OPEN READING FRAMES (ORFs) WITH YEAST GENEPAIRS® PRIMERS FROM RESEARCH GENETICS FOR MICROARRY EXPERIMENTS
PCR Amplification of Yeast Open Reading Frames (ORFs) with Yeast GenePairs® Primers from Research Genetics for Microarray Experiments
DNA microarrays comprise an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide or other solid substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the DNA on the microarray. This protocol describes the amplification of Saccharomyces cerevisiae genes and preparation of the amplified DNA for spotting on coated slides. Procedure
A. PCR Amplification
1. Sets of primers corresponding to the entire set of ORFs within the yeast genome (6,144 ORFs) are supplied in a 96-well microtiter plate format. Transfer 2 μl from the stock primers to fresh 96-well plates.
2. Prepare the following cocktail for each 96-well plate:
1000 μl of 10X PCR Buffer (Perkin Elmer)
800 μl of 25 mM Magnesium Chloride
100 μl of 100X dNTPs
20 μl of 0.2 mg/ml Yeast genomic DNA
7.41 ml of ddH2O
35 μl of AmpliTaq™ DNA Polymerase
Mix gently by trituration
3. Add 88 μl of the cocktail to each well of the 96-well plates that contains primers.
4. Place the plates in thermocycling machines and carry out polymerase chain reaction (PCR) with the following conditions:
36 cycles of:
92°C for 30 sec
56°C for 45 sec
72°C for 3 min 30 sec
B. Gel Electrophoresis of PCR Reactions for Quality Control
1. Transfer the PCR products to fresh 96-well microtiter plates that have U-shaped wells (Costar). Leave behind 3 μl of the PCR products in the original plates.
2. For each plate, pour a large 1% (w/v) Agarose Gel with four combs of 26 wells per comb.
3. Add 1 μl of 6X Loading Dye to each well that contains 3 μl of the PCR reactions.
4. Load 6 μl of DNA Size Ladder in the first well ("lane 1") of each row of wells (4 total) on the Agarose gel.
5. Using a 12-channel pipettor, load samples A1 to A12 into alternating even-numbered lanes (i.e., 2, 4,..., 24; see Hint #1).
6. Load samples B1 through B12 into alternating odd-numbered lanes (i.e., 3, 5,..., 25).
7. Repeat this procedure for the remaining samples such that two sequential rows of PCR reactions from the microtiter plate are loaded into a single row of wells in alternating lanes on the Agarose Gel.
8. Electrophorese the gel at 70 to 80 Volts until the first dye band (BPB) is equidistant between the originating wells and the next row of wells in the gel (see Protocol ID#1265).
9. Take high (open shutter for 1 sec) and low (open shutter for 6/30 of a second) exposure photographs of the gel with ultraviolet (UV) light illumination. Compare the molecular weights of the bands in the lanes to the predicted ORF sizes and look for the presence of significant "doublets"--two bands instead of one.
10. Repeat PCR reactions for the ORFs that did not amplify (see Hint #2).
C. DNA Precipitations
1. Lyophilize the samples in the "U-bottom" plates from Step #B1 to a volume to approximately 50 μl (see Hint #3).
2. Add 0.1 volumes of 3 M Sodium Acetate, pH 5.2, and 2.5 volumes of 100% Ethanol. Store the plates at -20°C for a few hours to overnight.
3. Centrifuge the plates in a Sorvall™ RC-3B centrifuge at 3500 rpm (3,565 X g) for 1 hr with the H-6000A rotor.
4. Remove the supernatant from each well with a 12-channel aspirator (Wheaton/PGC Scientifics).
5. Add 100 μl of ice-cold 70% (v/v) Ethanol to each well and centrifuge again for 30 min.
6. Dry the pellets under vacuum for 10 min.
7. Resuspend the DNA in 100 μl of ddH2O. Incubate the plates overnight at room temperature.
8. Transfer each sample in 10 μl aliquots to 384-well plates (USA Scientific or Corning Costar). These are the "print" plate sets.
9. Dry down the samples in the print plate sets under vacuum. Tightly seal the plates with aluminum foil (R.S. Hughes) for long-term storage at room temperature.
10. Before use, resuspend one print set in 4 μl of 3X SSC overnight at room temperature.
11. Spot the resuspended DNA from each plate onto Polylysine-coated slides (see Protocol ID #2258) with a 16-tip or 32-tip Arrayer. Dry down the used print plates for storage until next use.
SSC (20X) pH 7.2
3 M NaCl
0.3 M Sodium Citrate
AmpliTaq™ DNA Polymerase (Perkin Elmer) 5 Units/μl Taq DNA Polymerase 10X PCR Buffer (Perkin Elmer) Supplied with Taq DNA Polymerase dNTPs (100X) 25 mM each: dATP, dCTP, dTTP, dGTP 3 M Sodium Acetate, pH 5.2 25 mM Magnesium Chloride 25 mM MgCl2 70% (v/v) Ethanol DNA Size Ladder 1 ml of 6X Loading Dye
4 ml of 1X TE buffer
1 ml of 1 kb DNA Fragment Ladder (Gibco/BRL)
Loading dye (6X) 0.25% (w/v) Xylene Cyanol FF (XCF)
15% (w/v) Ficoll 400
0.25% (w/v) Bromophenol Blue (BPB)
Buffer 1X TAE
0.5 mg/ml Ethidium Bromide (CAUTION! See Hint #4)
Agarose Gel 1X TAE
1% (w/v) Agarose
0.5 mg/ml Ethidium Bromide (CAUTION! See Hint #4)
BioReagents and Chemicals
DNA Polymerase, AmpliTaq
Xylene Cyanol FF
1. The 96-well microtiter plate will have 12 columns (represented by numbers) and 8 rows (represented by the alphabet). Therefore, each of the 96 wells can be identified by a unique column and row designation (e.g., A12, C8, etc.).
2. For the second PCR attempt, sort the failures by the following criteria:
differences in gene size
Modify reaction conditions accordingly (see Protocol ID#1242).
For genes that still produce PCR failures, design new primers (e.g., to amplify subregions of genes instead of the whole gene).
3. The drying will be uneven, with wells around the edges experiencing more evaporation than the wells towards the center of the plate; however, 1 hr results in all the wells at or below 50 μl.
4. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
Citation and/or Web Resources
1. Lab website