This experiment describes the method of gel purification. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

Gel purification experiments

Materials and Instruments

Distilled Water Ethanol Formamide Gum Buffer Methylene Blue Sodium Dodecyl Sulfate Storage Solution TAE Buffer Tris Acetic Acid Acetate Na EDTA Blue Dextran TEN Buffer 40mer Polyacrylamide Gum
Scalpel UV Lamp Safety glass for UV filtration

Move

I. Materials

1. Buffers, solutions and reagents

Distilled water

Ethanol, 95%

Formamide (remove ions with Amberlite Single Bed Ion Exchange Resin MB-1 and store in the dark)

The buffer for the gel is standard TBE + 6mol/L urea

Methylene Blue, 100X, 0.05%

Sodium dodecyl sulfate storage solution, 10%

TAE Buffer

40 mmol/L Tris acetate

20 mmol/L Na acetate

0.2 mmol/LEDTA,pH8.3

Blue dextran, 1 mg/ml

TEN buffer

10 mmol/L Tris-HCl, pH 7.9

10 mmol/LNaCl

0.1 mmol/LEDTA

2. Nucleic acids and oligonucleotides

40mer (40bp oligonucleotide)

3. gels

Polyacrylamide gel where the ratio of acrylamide: bisacrylamide is 100:1 (total 12% acrylamide) and the size of the gel is 40 cmX20 cmX2 mm

4. special equipment

Dissecting knife

UV lamp (UVGL-58, Ultraviolet product)

UV-filtered safety glass

II. Methods

1. Suspend each 40mer in TEN buffer at a final concentration of 1mmol/L and sample only 1000pmol (1ul+4ul formamide) per lane (1cm wide).

2. When the 40mer has migrated about 30-35 cm, the gel is immersed in distilled water and the water is changed twice to remove the urea, and then stained overnight with 0.0005% methylene blue. The visible blue bands do not migrate accurately and synchronously, due to sequence effects, even though they are all 40bP oligonucleotides. Another alternative to staining is to detect the bands in the gel by UV projection, where a handheld UV light source is used to illuminate the gel on plain printer paper from above, wearing goggles, and the fluorescence of the bands in the limb is weaker than that of the surrounding fluorescence.

3. Cut off the predominant band of each species with a new scalpel and soak for 1 or 2 nights at 37°C in 3 times the volume of 0.1% SDS and TAE. Recover the supernatant (to avoid breakage of the gel) and precipitate with 3x volume of ethanol.

4. Estimate the yield of 40mer after reelectrophoresis and methylene blue staining by comparing it with some added unpurified 40mer as representative data for all oligonucleotides. In the experiment, the overall yield was approximately 30%, and this data was used for all oligonucleotides.

5. The oligonucleotides were pooled and the concentration of each was adjusted in the TEN buffer to

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