Focus: DNA Purification

Removal of Ethidium Bromide and Calf Intestinal Alkaline Phosphatase Using the Wizard^® SV Gel and PCR Clean-Up System

By Megan Buros and Natalie Betz, Ph.D.
Promega Corporation

Introduction

Many common laboratory reagents, such as calf intestinal alkaline phosphatase (CIAP) and ethidium bromide, can have detrimental effects on downstream DNA applications or can be potential health hazards. The enzyme CIAP is often used to dephosphorylate vector DNA fragments to prevent religation. However, failure to remove CIAP prior to ligation can reduce the efficiency due to the removal of the 5´ phosphate group from the insert DNA. Since successful ligation requires that at least one of the DNA fragments possesses a 5´ phosphate group, dephosphorylated insert DNA can no longer ligate to the dephosphorylated vector and is functionally removed from the reaction. Like CIAP, the DNA stain ethidium bromide can interfere with downstream applications (1). Ethidium bromide is also a known mutagen and requires special handling and disposal procedures. These experiments were conducted to determine if CIAP and ethidium bromide would interfere with DNA purification using the Wizard^® SV Gel and PCR Clean-Up Systems and if these reagents were removed effectively.

Methods

Removal of Calf Intestinal Alkaline Phosphatase: One unit of Calf Intestinal Alkaline Phosphatase (Cat.# M1821) was added to 50μl of a 500bp PCR product. Replicate samples of the PCR product with or without CIAP were purified using the Wizard^® SV Gel and PCR Clean-Up System or by phenol/chloroform extraction followed by ethanol precipitation. For the Wizard^® System, the DNA was eluted with 50μl of Nuclease-Free Water (Cat.# P1193). For the phenol/chloroform extraction and ethanol precipitation, the DNA pellet was resuspended in 50μl Nuclease-Free Water. The unpurified PCR product without CIAP, unpurified PCR product with CIAP, or purified PCR products were then ligated into the pGEM^®-T Easy Vector using the pGEM^®-T Easy Vector System (Cat.# A1360). Ligation of purified PCR products were performed in triplicate, as described in the pGEM^®-T Easy Vector System Technical Manual #TM042. Chemically competent JM109 cells were transformed with 2μl of the ligation reaction and plated onto LB plates containing IPTG, X-gal, and 125μg/ml ampicillin. After overnight growth at 37°C, colonies were counted. Light blue colonies were counted as white colonies since both types were determined to have insert DNA.

Removal of Ethidium Bromide: In a separate experiment, 5μl of Ethidium Bromide (Cat.# H5041) and 20μl of water were added to 25μl of the 1kb DNA Ladder (Cat.# G5711). Two replicates of 50μl each were purified using the Wizard^® SV Gel and PCR Clean-Up System as described in the Wizard^® SV Gel and PCR Clean-Up System Technical Manual #TB308. Equivalent volumes of the 1kb DNA Ladder before and after purification were separated on a 2% agarose gel and visualized on a UV transilluminator. Photographs were taken before and after ethidium bromide staining of the gel.

Results

Table 1 shows the results of experiments designed to examine the effectiveness of CIAP removal. Purified or unpurified PCR products were ligated into the pGEM^®-T Easy Vector. A ligation with vector but no PCR product was performed as a negative control, and the Control Insert DNA supplied with the pGEM^®-T Easy Vector System was used as a positive control for ligation. The number of white colonies, which represent plasmid DNA with insert, is dramatically lower with the unpurified PCR products as compared to those with purified PCR products, regardless of the purification method. Ligation of PCR products purified using the Wizard^® SV Gel and PCR Clean-Up resulted in more colonies than phenol/chloroform extraction and ethanol precipitation but gave comparable results for the percentage of colonies that contain insert. Similar results were obtained with DNA purified using the Wizard^® SV 96 PCR Clean-Up System (Cat.# A9340, data not shown).

Table 1. Ligation results of unpurified or purified PCR products.
Sample	Dark Blue Colonies	White Colonies	Colonies with Inserts
No insert	70	0	0%
pGEM®-T Easy Control Insert DNA	120	1352	92%
Unpurified PCR Product –CIAP	112	228	67%
Unpurified PCR Product +CIAP	17	119	88%
Phenol/Chloroform Extraction and Ethanol Precipitation	91	1071	92%
Wizard® SV Gel and PCR Clean-Up System	175	1619	90%

To monitor ethidium bromide removal, purified and unpurified 1kb DNA Ladder were visualized on an agarose gel before and after ethidium bromide staining. Prior to staining, no DNA bands are visible on the gel for the purified 1kb DNA Ladder, indicating removal of the ethidium bromide (see Figure 1, Panel A). To confirm the presence of DNA, the gel was then stained with ethidium bromide and visualized again. DNA fragments are clearly visible (see Figure 1, Panel B). The staining intensities are equivalent for DNA samples before and after purification, indicating efficient recovery of DNA from solutions containing ethidium bromide.

Conclusion

The Wizard^® SV Gel and PCR Clean-Up System effectively remove both calf intestinal alkaline phosphatase and ethidium bromide from DNA solutions. DNA purified with this system gave comparable ligation efficiency to that of DNA purified by phenol/chloroform extraction and ethanol precipitation. The Wizard^® SV Gel and PCR Clean-Up System does not involve the use of hazardous chemicals, unlike phenol/chloroform extraction and ethanol precipitation.

References

1. Aktipis, S. and Panayotatos, N. (1976) Mechanism of ethidium bromide inhibition of RNA polymerase. Biochem. Biophys. Res. Comm. 68, 465–70.

Products may be covered by pending or issued patents or may have certain limitations on use. Please visit our patent and trademark web page for more information.

pGEM and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office.

©2008 Promega Corporation. All rights reserved. | Patents, Trademarks and Product Usage Info | Privacy Policy | Contact Us

Customer Service: 608-274-4330 | Technical Service: 608-274-4330