Improved recovery of DNA from polyacrylamide gels after in situ DNA footprinting.
نویسندگان
چکیده
Methods used to date for the isolation of DNA from polyacrylamide gels are elution based, time-consuming and with low yield in DNA. This paper describes an improved system employing polyacrylamide gels made of a meltable matrix. The new system was successfully applied to in situ DNA footprinting following gel retardation assays.
منابع مشابه
Solid phase technology improves coupled gel shift/footprinting analysis.
For the analysis of protein-DNA interactions by coupled gel-shift/footprinting, DNA fragments need to be extracted from polyacrylamide gels and subsequently separated on high resolution gels. Due to impurities in the extracted DNA, single nucleotide resolution is frequently not achieved. We now describe an improved experimental strategy that employs transient coupling of DNA fragments to a soli...
متن کاملUsing a Temperature Gradient against the Time in Polyacrylamide Gel Electrophoresis May Eliminate the Need for Stacking Gels
Background and Objectives: Making stacking gels for polyacrylamide gels in the laboratory by conventional methods is laborious and time consuming. Considering the role of temperature in polyacrylamide gels with respect to electrical resistance and viscosity, we assumed that decreasing the temperature would cause an increase in electrical resistance and viscosity. Ultimately, a downward tempera...
متن کاملFluorescence-based protein footprinting using histidine-tagged protein.
We describe a procedure for protein footprinting to identify the region(s) of a protein that interacts with a ligand. The method utilized the affinity of a stretch of histidine residues cloned into the protein to metal-chelated resin. After limited protease digestion, the histidine-tagged end fragments were separated by the resin and labeled with a fluorescein derivative. Resolving the labeled ...
متن کاملFootprinting with an automated capillary DNA sequencer.
Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescei...
متن کاملSequence-specific binding of luzopeptin to DNA.
We have examined the binding of luzopeptin, an antitumor antibiotic, to five DNA fragments of varying base composition. The drug forms a tight, possibly covalent, complex with the DNA causing a reduction in mobility on nondenaturing polyacrylamide gels and some smearing of the bands consistent with intramolecular cross-linking of DNA duplexes. DNAase I and micrococcal nuclease footprinting expe...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Journal of microbiological methods
دوره 54 2 شماره
صفحات -
تاریخ انتشار 2003