DNA重组

Overview An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average every DNA molecule i ...

ProcedureA. Restriction Digest 1. Cut 10 to 15 μg of CsCl-purified plasmid DNA containing the fragment of interest with 2-fold excess of Restriction Enzyme for 2 hours (see Hint #2 and #3). 2. Add an ...

ProcedureA. Restriction Digest 1. Cut 10 to 15 μg of CsCl-purified plasmid DNA containing the fragment of interest with 2-fold excess of Restriction Enzyme for 2 hours (see Hint #2 and #3). 2. Add an ...

FootprintingFootprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds. Examples: hormone-receptor complexes that bind to their hormone response el ...

DNaseI FootprintintSolutions10X Binding Buffer200 mM Tris 8.0 200 ml 1M Tris pH 8.0500 mM NaCl 100 ml 5M NaCl10 mM EDTA 20 ml 0.5 M EDTA pH 8.0680 ml Qstore at room temperatureDNaseI Dilution Buffe ...

Author: Long-Cheng LiSource: Protocol OnlineAbstract: Simplified method for preparing G+A ladder run along with footprinting reaction. It's much simple than the original Maxima-Gilbert sequencing rea ...

DNaseI FootprintintSolutions10X Binding Buffer200 mM Tris 8.0 200 ml 1M Tris pH 8.0500 mM NaCl 100 ml 5M NaCl10 mM EDTA 20 ml 0.5 M EDTA pH 8.0680 ml Qstore at room temperatureDNaseI Dilution Buffe ...

Author: Long-Cheng LiSource: Protocol OnlineAbstract: Simplified method for preparing G+A ladder run along with footprinting reaction. It's much simple than the original Maxima-Gilbert sequencing rea ...

Principles of nucleic acid hybridization5.2.1. Nucleic acid hybridization is a method for identifying closely related nucleic acid molecules within two populations a complex target population and a co ...

Preparation of nucleic acid probesIn standard nucleic acid hybridization assays the probe is labeled in some way. Nucleic acid probes may be made as single-stranded or double-stranded molecules (see F ...

Nucleic acid hybridization assays using cloned target DNA and microarray hybridization technologySome of the technologies described in the preceding section (e.g. Southern blot hybridization and dot-b ...

Nucleic acid hybridization assays using cloned DNA probes to screen uncloned nucleic acid populationsNumerous applications in molecular genetics involve taking an individual DNA clone and using it as ...

Nucleic acid hybridization assays using cloned DNA probes to screen uncloned nucleic acid populationsNumerous applications in molecular genetics involve taking an individual DNA clone and using it as ...

In a microcentrifuge tube prepare a solution of linear DNA (25-50ng) in deionized water or TE buffer (10-35µl). Add: 10X ligation buffer 5µl 50% PEG 4000 solution (for blunt ends only) 5µl deionized ...

DNA labeling by nick translation reagents: DNA for labeling (concentration c 150 ng/µl) modified nucleotides: Biotin-16-dUTP Digoxigenin-11-dUTP conc. 1nmol/µl (Boehringer Mannheim) dNTPs (regular nu ...

DNA labeling by nick translation reagents: DNA for labeling (concentration c 150 ng/µl) modified nucleotides: Biotin-16-dUTP Digoxigenin-11-dUTP conc. 1nmol/µl (Boehringer Mannheim) dNTPs (regular nu ...

Linker Ligation (with T4 DNA Ligase) In a microcentrifuge tube prepare a solution of blunt ended dephosphorylated DNA (100-500ng) in TE buffer (5-7µl). Add 1-2µg of phosphorylated linkers in 5µl of TE ...

De-phosphorylation of DNAThe preferred method of de-phosphorylation uses the buffer system described by Pharmacia for most of their restriction endonucleases. You will need: 10 x OPA+ Buffer (100mM Tr ...

Thermal InactivationA simple reversible way to a stop restriction reaction is by adding EDTA which chelates Mg2+ thereby preventing catalysis. If further manipulations of the digested DNA are to be pe ...

The restriction/modification system in bacteria is a small-scale immune system for protection from infection by foreign DNA. W. Arber and S. Linn (1969) Plating efficiencies of bacteriophage lambda (l ...