PCR產物純化方法

分析測試百科網 > 行業資訊 > 技術原理

PCR產物純化方法

2019.8.08

Purification of PCR Products in Preparation for Cloning

Joseph Sambrook Peter Maccallum Cancer Institute and The University of Melbourne, Australia David W. Russell University of Texas Southwestern Medical Center, Dallas Excerpted From Molecular Cloning: A Laboratory Manual Third Edition

ABSTRACT
The residual enzymatic activity of thermostable DNA polymerases that survive the rigors of PCR can compromise subsequent enzymatic reactions. This protocol describes how to use proteinase K to destroy thermostable enzymes and to purify amplified DNA in preparation for cloning.

MATERIALS
Reagents and Solutions
Ammonium acetate (10 M) Amplified DNA from polymerase chain reactions Chloroform Ethanol Phenol:chloroform (1:1, v/v) TE (pH 8.0)
Enzymes and Buffers Proteinase K (20 mg/ml) Genomic grade proteinase K that has been shown to be free of DNase and RNase activity.
Gels EthanolAgarose gel containing 0.5 μg/ml ethidium bromide

METHOD
Pool up to eight PCRs (400 μl) containing 1 μg of the desired amplification product. (See Troubleshooting.) If mineral oil was used to prevent evaporation during PCR, centrifuge the pooled samples briefly and transfer the lower (aqueous) phase to a fresh microcentrifuge tube. Add 0.2 volume of 5x proteinase K buffer and proteinase K to a final concentration of 50 μg/ml. Incubate the mixture for 60 minutes at 37°C. Inactivate the proteinase K by heating to 75°C for 20 minutes. Extract the reaction mixture once with phenol:chloroform and once with chloroform. Add 0.2 volume of 10 M ammonium acetate and 2.5 volumes of ethanol. Mix the solution well and store it for 30 minutes at 4°C. Recover the DNA by centrifugation at maximum speed for 5 minutes at 4°C in a microcentrifuge. Discard the supernatant and then wash the pellet with 70% ethanol. Centrifuge again, remove the supernatant, and then allow the DNA to dry. The DNA may be further purified by chromatography or by gel electrophoresis. This step is recommended when primers have been used to add restriction sites to the ends of the amplified DNA. Unused primers and primer-dimers should be removed before digesting the DNA with the appropriate restriction enzymes (please see Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration). Dissolve the pellet in TE (pH 8.0). Assume that the recovery of amplified DNA is 50-80% and dissolve the DNA in TE (pH 8.0) at an estimated concentration of 25 μg/ml (25 ng/μl). Analyze approx. 25 ng of the purified DNA by agarose-ethidium bromide gel electrophoresis, using markers of an appropriate size. Check that the amplified band fluoresces with the intensity expected of approx. 25 ng of DNA.
TROUBLESHOOTING
Nonspecific amplification products are present at significant levels (e.g., are detectable by gel electrophoresis). Step 1 Purify the desired product by electrophoresis through low-melting-temperature agarose before proceeding (please see Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction).

REFERENCES
Crowe J.S., Cooper H.J., Smith M.A., Sims M.J., Parker D., Gewert D. 1991. Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion. Nucleic Acids Res. 19: 184-184.[Free Full Text] Wybranietz W.A. and Lauer U. 1998. Distinct combination of purification methods dramatically improves cohesive-end subcloning of PCR products. BioTechniques 24: 578-580.[Medline]

Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult: Molecular Cloning: A Laboratory Manual Third Edition, by Joseph Sambrook and David W. Russell, ? 2001 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. . 8.25-8.26.

Copyright ? 2006 by Cold Spring Harbor Laboratory Press. All rights reserved. No part of these pages, either text or image may be used for any purpose other than personal use. Therefore, reproduction modification, storage in a retrieval system or retransmission, in any form or by any means, electronic, mechanical, or otherwise, for reasons other than personal use, is strictly prohibited without prior written permission

生物在線

喜歡作者我要約稿

PCR產物純化方法

NGS上Hiseq機器前的準備工作：核酸提取、文庫構建、簇生成

瓊脂糖凝膠電泳回收PCR產物

PCR產物的直接純化原理、材料與方法

測序常見問題及其分析

測序常見問題及其分析

以硅膜為基礎純化 PCR 產物實驗方法

PCR 產物純化實驗

制備克隆用PCR產物的純化

測序常見問題及其分析

pcr產物的純化與回收實驗報告

測序常見問題及其分析（圖）

DNA小片段的純化回收

PCR產物的直接純化原理、材料與方法

以硅膜為基礎純化 PCR 產物實驗方法

PCR 產物純化實驗

PCR產物純化方法

PCR產物常規純化方法

以硅膜為基礎純化 PCR 產物實驗方法

PCR 產物純化實驗

制備克隆用PCR產物的純化

周錦帆

喜歡作者

打賞方式

PCR產物純化方法

周錦帆