DNA基础知识

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 ...

Partial Endonuclease Digestion Prepare a 100 µl reaction mixture containing the DNA of interest in the appropriate 1X restriction enzyme buffer. Divide the reaction mixture between five prechilled mic ...

Restriction digestionRestriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme in its respective buffer as recommended by ...

Restriction enzymes are expensive ($40 to $200 a vial): keep the enzymes at -20 C. Plan your digests to be small and convenient. The digest is composed of DNA sample (volume should not be more than a ...

Restriction digestionRestriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme in its respective buffer as recommended by ...

Materials: Restriction enzymes of choice such as BamH1 and EcoRI Restriction enzyme reaction buffer such as MULTI-CORE (TM) (Promega) 70 % Ethanol 100 % Ethanol 3 M Sodium acetate (pH 5.2) Distilled w ...

Restriction Enzyme Buffer Most enzymes can use REact buffers; however some are made up separately. Use fresh Milli-Q water siliconized or sterile glassware or disposable plastic ware to make the follo ...

Materials: 10X restriction enzyme buffer (see manufacturer's recommendation) DNA sterile water restriction enzyme phenol:chloroform (1:1) Add the following to a microfuge tube: 2 μl of appropriate 10X ...

Terminal Deoxynucleotidyl TransferaseTerminal Deoxynucleotidyl Transferase (TdT) is an enzyme that catalyzes the repetitive addition of mononucleotides from dNTPs to the terminal 3´-OH of a DNA initia ...

Genomic DNA Labeling ProtocolWe typically use 0.5ug of E. coli genomic DNA in a labeling reaction for each hybridization. The genomic DNA was fragmented to 500 to 1000bps before labeling. The followin ...

Fill-in Labeling of DNA FragmentsThis protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA ...

Fill-in Labeling of DNA FragmentsThis protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA ...

Typical 5'-kinase labeling reactions included the DNA to be labeled -32-P-rATP T4 polynucleotide kinase and buffer (3). After incubation at 37degC reactions are heat inactivated by incubation at 80deg ...

Labeling oligonucleotides with 32P ATPSteve HahnLast modified 8/13/01Wear gloves throughout and work in radiation area. Monitor area before and after use.Mix the following in an eppendorf tube:1. 0.5 ...

Spin Column chromatographyUsed to removed unincorporated nucleotides from labelling reactions.- Prepare Sephadex G-50 (medium) by adding appropriate amount of dry beads to 100 ml TE buffer such that t ...