DNA重组

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 ...

DESCRIPTIONLabeling of DNA by random oligonucleotide-primed synthesis is based on the investigations of A.Feinberg and B.Vogelstein 1 2 and is a good alternative to nick translation for producing uni ...

RADIOLABELING OF PROBES FOR ELECTROPHORETIC MOBILITY SHIFT ASSAYS ...

DNA probes are prepared using a modification of the method of Feinberg and Vogelstein (1983). You will need: Nuclease-free BSA-dCTP (Amersham or DuPont)Klenow DNA Polymerase (New England Biolabs or Ph ...

DNA probes are prepared using a modification of the method of Feinberg and Vogelstein (1983). You will need: Nuclease-free BSA-dCTP (Amersham or DuPont)Klenow DNA Polymerase (New England Biolabs or Ph ...

IntroductionThe efficiency of nucleotide incorporation in DNA/RNA polymerization reactions (e.g. transcription reverse transcription and DNA replication) can be determined by trichloroacetic acid (TCA ...

IntroductionThe efficiency of nucleotide incorporation in DNA/RNA polymerization reactions (e.g. transcription reverse transcription and DNA replication) can be determined by trichloroacetic acid (TCA ...