4. Perform the following thermal cycle series: 1 initial cycle of heating to 96°C and holding for 30 sec; 25 cycles of denaturing at 94° C for 30 sec, reannealing at 55°C for 30 sec, and extending at 72°C for 150 sec; one final cycle of holding at 72°C for 5 minutes, then cooling to ambient temperature.
After PCR, plates made be held at 4°C while quality controls are performed.
2.4 Check PCR products by agarose gel electrophoresis of ESTs
If this is the first time the template for these ESTs is being amplified, analyze 2 µl of each PCR product on a 2% agarose gel. If amplified products from this template have been previously tested, then analyze one row of wells from each plate amplified. Gel imaging allows a rough quantitation of product while giving an excellent characterization of the product. Band size, as well as the number of bands observed in the PCR products, contribute to understanding the final results of the hybridization. The use of gel well formats suitable for loading from 96 well plates and programmable pipetters makes this form of analysis feasible on a large scale.
Materials, Reagents and Solutions
Electrophoresis apparatus with capacity for four 50 well combs, (e.g. #D3, Owl Scientific, Woburn, MA)
50 X Tris-Acetate Electrophoresis BufferM
Agarose
Dye Solution (Xylene Cyanol/Bromophenol Blue) (e.g. #351-081-030, Quality Biological Inc., Gaithersburg MD)
Glycerol (enzyme grade)
Ethidium Bromide solution (10 mg/ml)
100 base-pair ladder size standard
Programmable, 12-channel pipetter (e.g. #2019, Matrix Technologies, Lowell, MA)
Disposable microtiter mixing trays (e.g. Falcon #353911, Becton Dickinson, Franklin Lake, NJ)
Electrophoresis power supply
1X TAE Buffer
50X TAE Buffer 40 ml
Ethidium Bromide (10 mg/ml) 0.1 ml
Water 960 ml
1000 ml
Loading Buffer
Glycerol (enzyme grade) 4.0 ml
DEPC Water 0.9 ml
Dye Solution* 0.1 ml
5.0 ml
(*This solution is 0.25% (w/v) Xylene Cyanol and 0.25% (w/v) Bromophenol Blue)
100 bp Size Standards
DNA ladder (1 mg/ml) 50 µl
1 M Tris-HCl (pH 8.0) 5 µl
0.5 M EDTA (pH 8.0) 5 µl
Loading Buffer 440 µl
500 µl
Method
1. Cast a 2% agarose gel (1X TAE) with four combs (50 tooth) and submerge in an electrophoresis apparatus with sufficient 1X TAE buffer to just cover the surface of the gel.
2. Prepare a reservoir of Loading Buffer, using 12 wells of a microtiter plate.
2. Program pipetter to sequentially carry out the following steps:
fill with 2 µl
fill with 1 µl
fill with 2 µl
mix a volume of 5 µl five times
expel 5 µl
3. Place 12 disposable tips on the pipetter.
4. Load 2 µl of PCR product from wells A1-A12 of the PCR plate.
5. Load 1 µl of air.
6. Load 2 µl of Loading Buffer from the reservoir.
7. Place tips in clean wells of disposable mixing tray and allow pipette to mix the sample and loading dye.
8. Place the pipette tip in a 50 well row so that the tip containing the PCR product from well A1 is in the second well of the row, and the other tips are in every other succeeding well.
9. Repeat the process (changing tips each time), loading PCR plate row B starting in the 3rd well, interleaved with the A row, the C row starting at well 26, and the D row at well 27, interleaved with the C row.
10. Place 5 µl of 100 bp Size Standards, in wells 1 and 50.
11. Repeat this process, loading samples from rows E, F, G, and H in the second, 50 well row of gel wells, loading samples from two 96 well PCR plates per gel, or single row samples from 16 PCR plates.
To reduce diffusion and mixing, apply voltage to the gel for a minute between loading each well strip. This will cause the DNA to enter the gel, and reduce band spreading and sample loss.
12. Apply voltage to the gel and run until the bromophenol blue (faster band) has nearly migrated to the next set of wells.
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