Agarose gel electrophoresis is used extensively to separate and analyze DNA that is subsequently used for downstream applications. Eppendorfs Perfectprep Gel Cleanup kit is used to purify DNA fragments of various sizes (70 bp10 kb) from agarose gels with recovery rates of up to 96%. Since the majority of DNA purified from agarose is to be used in other applications, it is critical that the isolated DNA is of high quality. One downstream application in which the DNA may be used is a restriction enzyme digest. Restriction enzyme digests are sensitive to inhibitors and are therefore good indicators of possible contaminants in purified DNA.
The Perfectprep Gel Cleanup kit was used to purify a 3.49 kb PCR1 fragment amplified from bacteriophage Lambda. The purified DNA was subjected to restriction enzyme digestion using EcoRI, EcoRV and HindIII. The digests were done in duplicate, and for comparison, the PCR product was also purified using a competitors kit and then subjected to the same restriction enzyme digests. In the EcoRV and HindIII digests, the DNA purified with the Eppendorf kit was cut as well as or better than the DNA isolated using the competitors kit. In all cases using EcoRI, the DNA purified with the Eppendorf kit was digested more effectively than was the DNA isolated using the competitors kit. This indicates that DNA obtained from agarose gels using the Eppendorf Perfectprep Gel Cleanup kit can be used in restriction enzyme digests, and the performance in these dig ests is better than the DNA obtained from the competitors kit.
A 3.49 kb fragment of Lambda DNA was amplified in 96 30 l PCR reactions using the following Eppendorf PCR reagents. (The numbers in parentheses represent the final amount or concentration of the reagent in each reaction.):
Template DNA (50 ng)
10 x Taq DNA Polymerase Buffer (1x)
dNTP Mix (0.2 mM)
25 mM MgCl2 (0.5 mM)
10 mM dNTP Mix (0.2 mM)
Taq DNA Polymerase (0.05 U/l)
Molecular Biology Grade Water
10 mM JMF1
5'-AAA-AAC-GTC-CGA-GAC-GAA-T-3' (0.1 mM)
10 mM JMrev3.6
5'-TCA-GCA-TGT-AGC-ATG-CAA-CC-3' (0.1 mM)
Integrated DNA Technologies (IDT)
The Lambda DNA was amplified using a Mastercycler gradient with the following
94C for 5 minutes
94C for 45 seconds
70C for 20 seconds
72C for 2 minutes
72C for 5 minutes (Final Polishing Step)
The samples were pooled after amplification.
20 l of the PCR sample were loaded into a 100 ml 1% SeaKem LE agarose gel containing 2 l of 10 mg/ml ethidium bromide. 18 lanes were loaded. The gel was run in 1x TBE at 117 V for 30 minutes.
Eighteen 3.49 kb bands were excised from the gel. One half of these were purified with the Eppendorf Perfectprep Gel Cleanup kit, and the remaining half were purified with the competitors kit. All samples were eluted in 30 l as described in the manufacturers protocols. Eluting the DNA fragments in the same volume gives an accurate comparison of the two kits. The final volume of the eluate was adjusted to 30 l using the appropriate elution buffer. Each sample was subsequently used for two restriction enzyme digests, ie, one duplicate.
10 and 20 units of EcoRI, EcoRV and HindIII were used to digest the purified PCR products. Master mixes were prepared for each enzyme such that 10 l of the master mix could be added to 15 l of the DNA sample for a total reaction volume of 25 l. The master mix was pipetted into the lid of the corresponding reaction tube. The lids were closed, and the tubes were briefly centrifuged to add the master mix to the DNA samples so that all of the reactions began at the same time. All reactions were incubated at 37C for 2 hours. The reactions were stopped by adding 2.5 l of 10x Gel Loading Buffer and then loaded onto a gel. The entire reaction volume (25 l) was run on a 150 ml 1.3% OmniPur agarose gel in 1x TBE at 95 V for 3 hours. The gel was stained in 50 ng/l ethidium bromide solution for 20 minutes and destained in deionized water for 30 minutes.
DNA purified from an agarose ge l using the Eppendorf Perfectprep Gel Cleanup kit can be successfully digested with restriction enzymes. In each trial of each enzyme, the DNA purified with the Eppendorf kit performed as well as or better than the DNA prepared with the competitors kit. Particularly, in both trials using EcoRI, the DNA obtained with the Perfectprep Gel Cleanup kit was cut markedly better than the DNA obtained with the competitors kit.