A). AGAROSE CONCENTRATIONS:
Use 0.8% agarose (w/v) for high molecular weight DNA fragments, and 1 - 1.2% for smaller DNA fragments. 0.5% gels are very flimsy and need to be handled with caution. Low melting agarose is used at the same concentration as regular agarose. We have used FMC's SeaPlaque agarose which melts at 65°C and solidifies @ about 30°C. We typically cut a trough in the regular agarose after electrophoretic separation and staining, pour the low melting temperature agarose in the trough, allow it to solidify, and then electrophorese the band into that agarose. The rigidity of low melting temperature agarose is low.
B). BUFFERS & ELECTROPHORETIC CONDITIONS:
Buffer choice: Both Helling's and TBE buffers are used for overnight runs. Use TBE for high voltage electrophoresis greater than 60 volts. The agarose may melt if Helling's buffer is used at high voltage. Resolution is higher for DNA fragments greater than 4 kb when using Helling's buffer, and in the lower molecular weight DNA range when using TBE. Elution of DNA from agarose with glass beads does not work with TBE.
Buffer Concentrations (diluted from concentrated stocks below):
2X Helling's buffer (large gels & IBI gel)
1X Helling's buffer (baby gels)
1X TBE buffer
C). Electrophoretic Conditions:
For quick electrophoretic separations, use 1X TBE at 50-100 volts for a few hours (baby gels 80cV 60 min). For overnight runs, use 40 constant volts for the large 12-20 square cm gels. For critical measurements, run the gel overnight at the lower voltage. High voltages lead to decreased resolution.
D). Gel Preparation:
Measure agarose and dilute buffer to appropriate concentration. Boil in an Ehrlenmeyer ml flask. Cool to 60-65°C before pouring onto a clean, leveled, glass plate or plastic tray surrounded by masking tape. Insert the comb parallel to the plate's edge, with the bottom of the teeth about 2mm above the plate. Multiple combs can be used on a gel to accommodate additional samples needing only short separation distances. Use a 2mm comb is used for larger (greater than 30µl) volumes of DNA. The thinner the comb, the higher the resolution. Pour the molten agarose solution at room temperature or in the cold room. Use a Pasteur pipette to push any air bubbles that form to the side. The gel will solidify in the cold room in about 20-25 minutes, or about 45 minutes at room temperature. The gel is opaque when solid. Remove the comb carefully or you may rip out the bottom of the wells. Transfer the gel to the electrophoresis chamber. If the gel won't be run for 1-2 hours, submerse it in running buffer.
E). Sample Preparation:
Heat DNA samples to 65°C, 10-15 minutes before loading to disassociate overhanging ends of molecules which may have re-annealed. Load 0.4 - 0.6 µg of cloned DNA per lane for analytical gels (depending on the number of expected bands) and up to 20 µg of genomic DNA per lane. Load molecular weight markers! Gently overlay with buffer, or alternatively, load samples through the buffer. Do not puncture the bottom of the wells.
F). Staining the Gel:
Stain gels with 1 µg/ml ethidium bromide in running buffer for 20-45 minutes depending on the gel thickness after electrophoresis. This is preferred to running gels with stain in them since DNA may migrate aberrantly with ethidium bromide. Large agarose gels stain in about 45 minutes; baby gels take about 15 minutes. Wear gloves when working with ethidium bromide, as it is carcinogenic and mutagenic. Dispose of the ethidium bromide solution in the decontamination carboy.
G). Photography:
Use 1 second, f4.5 when using the Fotodyne U.V. trans-illuminator for Polaroid type 57 film. A 3 minute exposure is necessary for type 55 film (with negative).
20X Hellings (1M Tris, 0.4 M sodium acetate, 0.04 M EDTA, pH 8.05)
| Chemical | For 4 liters | For 2 Liters |
|---|---|---|
| Tris | 484.4 g | 242.2 g |
| Sodium Acetate (anhydrous) | 131.25 g | 65.6 g |
| EDTA | 59.56 g | 27.8 g |
pH with glacial acetic acid to 8.05 (try about 60 ml initially, check pH and adjust as needed. QS to 4 liters (or 2 L) with distilled water. Autoclaving is optional.
10X TBE (1M Tris, 1M Boric Acid, 20mM EDTA, pH 8.3) 242.2 g Tris 123.66 g boric acid 14.89 g EDTA Adjust pH to 8.3. QS to 2 liters. Autoclave. (Biotechniques 10:182, 1991 claims that filtering up to a 20x TBE solution through 0.2 - 0.45µ cellulose acetate or cellulose nitrate filters prevents formation of precipitants during long-term storage. The solution may be reautocalved to dissolve precipitates that form.) Ethidium Bromide Stock Solution (10mM Tris-HCl, 1 mM EDTA, 1 mg/ml ethidium bromide) For 50 ml: 0.5 ml 1M Tris-HCl, pH 8.0 0.1 ml 0.5M EDTA 1 mg ethidium bromide Add ethidium bromide to Tris-HCl, EDTA and about half of the water, and stir overnight to dissolve. QS to 50 ml with water. Store in a brown bottle at room temperature.
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