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Method for recovering DNA from polyacrylamide after electro phoresis

A polyacrylamide gel and electrophoresis technology, applied in the field of DNA recovery, can solve the problems of low DNA recovery amount, long operation time, large DNA amount, etc., and achieve the effects of high purity, short operation time, and large recovery amount

Inactive Publication Date: 2007-03-28
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the shortcomings of existing conventional methods, such as long operating time and low DNA recovery, and to provide a method for recovering DNA from polyacrylamide gel after electrophoresis, which can recover DNA from polyacrylamide gel within 4 hours. The DNA of the target fragment is recovered from the gel, and the obtained DNA is large in quantity and high in purity, which can be used for subsequent re-amplification operations

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] DNA was recovered from the polyacrylamide gel after electrophoresis according to the following procedure:

[0021] 1) Cut out the gel strip containing DNA fragments with acrylamide concentration of 6% under ultraviolet light (the strip is 1 cm long, 2 mm wide, and 0.75 mm thick), and put it into a 1.5 ml centrifuge tube.

[0022] 2) Add 150μl ddH with a pipette 2 O Clean the strip three times, and when finished, suck out the cleaning solution.

[0023] 3) Grind the gel with a gel grinder until it becomes powdery.

[0024] 4) Add 150 μl of TE buffer, components: 15 mM Tris, 10 mM EDTA (pH 8.0). Rinse the grinding rod repeatedly until the grinding rod is free of gel.

[0025] 5) Water bath at 70°C for 2 hours, shaking several times during the period. Centrifuge at 8000 rpm for 2 minutes, and absorb the supernatant.

[0026] 6) Transfer the supernatant to a new centrifuge tube, add 450 μl of DNA ligation solution containing 6M GuSCN and 200 mM Tris, adjust the pH to 6...

Embodiment 2

[0033] DNA was recovered from the polyacrylamide gel after electrophoresis according to the following procedure:

[0034] 1) Cut out the strip containing the DNA fragment with an acrylamide concentration of 8% under ultraviolet light (the length of the strip is 0.8cm, the width is 1.5mm, and the thickness is 1mm), and put it into a 1.5ml centrifuge tube.

[0035] 2) Add 200μl ddH with a pipette 2 O (Sterilization) Clean the strip three times, and suck out the cleaning solution after completion.

[0036] 3) Grind the gel with a gel grinder until it becomes powdery.

[0037] 4) Add 200 μl of 20 mM Tris, 15 mM EDTA (pH 8.0) TE buffer solution, and repeatedly wash the grinding rod until the grinding rod is free of gel.

[0038] 5) Water bath at 70°C for 2 hours, shaking several times during the period. Centrifuge at 10,000 rpm for 2 minutes, and absorb the supernatant.

[0039] The supernatant was transferred to a new centrifuge tube, and 600μl of 8M GuSCN, 300mM Tris was adde...

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PUM

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Abstract

The invention relates to the method used to reclaim DNA from polyacrylamide glue after electrophoresis. It includes the following steps: washing the adhesive tape sample by dH2O, rubbing into powdery by gel rubbing stick, gel soaking, gel removing, DNA connecting, DNA absorbing, DNA washing, DNA elution. Its the advantages are that it has large DNA recovery quantity, high purity, short operation time, simple operation, no need for special device, lost cost; and it can be used in kit manufacturing and agarose gel DNA recovery.

Description

technical field [0001] The invention relates to a method for recovering DNA from polyacrylamide gel after electrophoresis. Background technique [0002] After polyacrylamide gel electrophoresis, in some cases, such as: cloning and sequencing of electrophoresis fragments, it is necessary to recover the electrophoresis DNA fragments. Because acrylamide is polymerized by C-C bonds, it is difficult to depolymerize after polymerization. Enzymes cannot be used to depolymerize it like agarose gel, so as to facilitate the release of DNA from the gel; in addition, polyacrylamide gels are stable even at high temperatures. It will not melt, so it cannot be melted at a higher temperature like low-melting point agarose gel, so as to facilitate the release of DNA from the gel. In conventional polyacrylamide gel electrophoresis, the concentration of the gel is usually 6-10%. At this concentration, the gel lattice formed by the crosslinking of the polyacrylamide gel is dense, which is also...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 罗鹏胡超群任春华王青柏
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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