The purity of DNA extracted from agarose gels can have a significant impact on downstream applications. DNA fragments extracted and purified using the are free of salts, ethidium bromide, protein, agarose and organics, thereby allowing successful results in downstream applications. The provides a method for extraction of DNA from agarose gels for subsequent use in PCR*, sequencing, labeling and hybridization. DNA can be extracted from low and high melting temperature agarose in TBE or TAE buffer. This method has been optimized for purification of DNA fragments from 70 bp to 10 kb. Gel purification and binding capacity experiments (Fig. 1) show over 80% recovery of DNA when samples are processed using the .Experiment # Ave. Yield (g)
This experiment was performed using pCMVb (Clontech). Approximately 10 g of plasmid was processed using the Eppendorf Gel Cleanup Kit and Brand Q. Yield recovered was determined based on A260 reading. This experiment was performed to test binding capacity and recovery.
The Polymerase Chain Reaction was carried out on a 2 kb fragment of pCMVb (Clontech, Palo Alto, CA, USA) purified using the Eppendorf Perfectprep Gel Cleanup Kit and Brand Q Gel Extraction Kit. Eppendorf PCR reagents were used in the amplification reaction. Primers were obtained from Integrated DNA Technologies (Coralville, IA, USA):
LacZ F1 (5'-GGAAAGCTGGCTGGAGTGCGATCTT-3')
LacZ R2 (5'-TCCCCAGCGACCAGATGATCACACT-3').
A final concentration of 40 pg/l template was added to each reaction. Cycling parameters specific to the prime rs were used.
Sequencing was performed on 5 l of purified PCR products in triplicate. and Brand Q's Gel Extraction Kit were used for extraction and purification of the DNA fragment. Sequencing was carried out on a 377 ABI Sequencer. The results were analyzed and Phred Q20 scores were determined.
Labeling and hybridization
Dot Blot analysis was used to determine the effectiveness of labeling and hybridizing a DNA fragment purified using the Eppendorf Perfectprep Gel Cleanup Kit. The PCR product was gel extracted and purified, then labeled using the Ambion (Austin, TX, USA) Psoralen-Biotin Labeling Kit. 20 to 0.002 ng of gel extracted PCR product was blotted onto positively charged nylon and probed with the Psoralen-Biotin labeled probe. The Ambion Brightstar BioDetect Kit was used for detection of the labeled probe.
Amplification worked robustly when using DNA purified from the Eppendorf Perfectprep Gel Cleanup Kit and Brand Q (Fig. 2). PCR is a sensitive downstream application that can be affected by contaminants resulting from gel extraction methods. Based on the results obtained there is no detectable inhibition of the amplification reaction.Figure 2: Amplification of gel extracted PCR product Lane 1 1 kb Marker Lanes 24 Brand Q Lanes 57 Eppendorf Lane 8 No Template Control Lane 9 1 kb Marker Sequencing Results Sequencing is a downstream application that is equally sensitive to impurities. The quality of sequencing is a good determinant of the purity of the DNA extracted and purified using the gel cleanup kits (Fig. 3). Fig. 3: Sequencing trace of a PCR product purified using the Eppendorf Perfectprep Gel Cleanup Kit Average Phred Q20 scores (n=3) Eppendorf 502.33 Brand Q 436.67 As shown, the average Phred Q20 scores for the triplicate samples are superior for the Eppendorf samples as compared to Brand Q, indicating highest levels of purity.
Labeling and hybridization Results
When using a PCR product extracted and purified using the Eppendorf Perfectprep Gel Cleanup Kit, Psoralen-Biotin labeling can be performed efficiently. A standard serial dilution is seen as would be expected with proper labeling and hybridization.Fig. 4: Labeling and hybridization of PCR product extracted and purified using the . Series from left to right: 20 to 0.002 ng of PCR product blotted on the membrane. Conclusions
The is a quick, easy way to extract
pure DNA from agarose gels. Downstream applications that are sensitive
to DNA impurities are very successful when using DNA purified with the
Eppendorf kit. In sequencing and % re
covery the Eppendorf kit out-performs
the competitor in downstream applications.
*This product is sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc. and Applied Biosystems.