DNA实验技术

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 ...

Prepare a ligation mix:Ligation Mix (2x)10x ligase buffer1.0 mldigested vector(0.1 mg/ml)1.0 mlH2O6.0 mltotal8.0 mldivide ligation mix into two Eppendorf tubesLigation Rxn InsertControlligation mix ...

Cohesive End Ligation1) The ligation mixture contains the following: vector DNA (~100 ng) insert DNA (equimolar or 2 or 3 X molar concentration of vector) water added to 18 µl 2) Heat the mixture at 4 ...

set up the following reaction:CIP Rxn H2O7.8 ml10x cip rxn buffer2.0 mlDNA(e.g; 3 kb vector; 0.2 mg/ml; 2 mg total)10.0 ml(1 u/ml) CIP0.2 mltotal20.0 mlincubate for 60 min at 37°C add 1.14 ml 0.2 M ...

DEPHOSPHORYLATION OF LINEARIZED DNAALKALINE PHOSPATASE(CIP) DIGESTION Digestion of protruding 5'-ends1. Precipitate digested DNA and resuspend in a 100ml of 1X CIP Buffer.2. Add 1U CIP for 100pmol 5'- ...

DEPHOSPHORYLATION OF LINEARIZED DNAALKALINE PHOSPATASE(CIP) DIGESTION Digestion of protruding 5'-ends1. Precipitate digested DNA and resuspend in a 100ml of 1X CIP Buffer.2. Add 1U CIP for 100pmol 5'- ...