Use the tables below corresponding to the AquaPor agarose of choice to determine the percentage gel to cast. ( LE = general purpose; LM = low melting; ES = extra strength; HR = high resolution).
Preparing an Agarose Gel
COMB SELECTION:
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Use a thin (< 1 mm) comb with wide teeth for the sharpest, best-resolved bands.
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Be certain the comb is cleaned scrupulously prior to use.
BUFFER SELECTION:
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Use 1X TBE for optimal resolution of DNA < 12 kb when the DNA will not be recovered.
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Use 1X TAE for the best separation of DNA from 12 kb to 50 kb, or for DNA < 12 kb if the DNA will be recovered from the gel.
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Use 1X Tris-Acetate (TAE without EDTA) if the DNA will be used for in-gel enzymatic processing.
CASTING A GEL:
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Add buffer (at room temperature) to a flask that is 2.5 - 4 times the volume of gel solution. Add a teflon-coated stir bar.
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Add AquaPor powder while stirring vigorously so the agarose is dispersed uniformly. Stir for 2 minutes to hydrate the agarose.
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Tare the flask and solution.
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Place in a microwave oven and heat at 100% power using 20-60 second intervals. Swirl gently between intervals to resuspend the agarose.
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Continue the cycle of heating and swirling until the agarose is completely dissolved (no visible particles are present).
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Add distilled water to return the solution to its initial weight and mix.
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Cool the solution to 50-60°C before pouring the gel. Pour the solution into the mold so as to dispense the entire amount in 30-60 seconds, without generating bubbles.
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After casting, chill the gel for 30 minutes prior to comb removal when using AquaPor LM, HR, and low (<1%) concentrations of AquaPor LE and ES. This will complete gelation, increase gel strength, and enhance DNA resolution.
Preparation of Denaturing Agarose Gels
A variety of denaturants can be used with agarose. Alkaline gels are most often employed with single stranded DNA, because pouring and handling such gels is nonhazardous, as well as convenient. However, because strong alkali will hydrolyze RNA, formaldehyde is used with RNA.
Preparing Alkaline Agarose Gels
At high temperatures, alkaline conditions will hydrolyze the Agarose polysaccharide chains. To prepare an alkaline gel, the agarose is first melted in water and cooled to near gelling temperature. Buffer concentrate is then added and the gel is poured.
GEL PREPARATION:
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Dissolve 1.2g of Agarose in 98ml of deionized water. Stir agarose well into cold water, until all clumps are broken up. Heat the suspension in a microwave until it is a clear and homogeneous solution. Allow to cool to 15°C above gelling temperature.
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Add 2ml of 50X alkaline gel buffer.
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50X Alkaline gel buffer:
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1.5M NaOH
50mM EDTA
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Pour gel as described in the protocol above for the preparation of standard agarose gels.
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Run gel in 1X Alkaline gel buffer.
SAMPLE PREPARATION:
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Mix samples with an equal volume of:
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60mM NaOH
2mM EDTA
20% Ficoll
0.06% Bromocresol Green
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Stock solution is: 2g Ficoll, 0.4ml 50X Alkaline buffer, 6mg Bromocresol Green, bring to 10 ml with deionized water.)
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Preparing Formaldehyde Agarose Gels (for RNA Analysis)
Note: RNA is subject to rapid degradation by RNase present in the environment. For optimal results, use water treated with DEPC.
GEL PREPARATION:
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Melt 1.2g Agarose in 87ml of DEPC water, by dispersing the agarose uniformly and heating in a microwave until all particles are dissolved.
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Bring the melted agarose to 60°C.
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Add 10ml 10X MOPS Buffer and 3ml 37% formaldehyde. FORMALDEHYDE IS VOLATILE AND TOXIC. WORK IN A HOOD FROM THIS POINT FORWARD.
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10X MOPS (per liter):
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0.2M MOPS pH 7 with NaOH
50mM Sodium Acetate
10mM EDTA
Use DEPC treated water and RNase free reagents
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Pour gel as described in the protocol above for the preparation of standard agarose gels. USE A FUME HOOD!
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Allow gel to set for 1 hour.
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Run gel in 1X MOPS Buffer.
SAMPLE PREPARATION:
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Mix 40µl sample buffer with 10µl sample, heat to 55°C 15 minutes.
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Sample Buffer:
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65% Formamide
22% Formalin (37% formaldehyde)
13% 10X MOPS
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Add 10µl of: 50% glycerol, 1mM EDTA, 0.3% each Bromophenol Blue and Xylene Cyanol.
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