T4-polynucleotide kinase

Kits and enzymes Superscript reverse transcriptase (Invitrogen), Maxiscript and Megascript in vitro transcription kits (Ambion, Austin, TX). Taq-DNA polymerase, T4-polynucleotide kinase, EcoRI and Hindlll restriction enzymes (Invitrogen). 

Treat the purified DNA fragment by T4 polynucleotide kinase at 37°C for 1 h, then purify the DNA fragment by ethanol precipitation. 

T4 polynucleotide kinase Bacteriophage T4 Phosphorylation of 5 -OH terminus of a polynucleotide (DNA or RNA)... 

Koch et a1.1401 developed a method in which a peptide sequence (H-Leu-Arg-Arg-Ala-Ser-Leu-Gly-OH, the Kemptide ) was synthesized either at the N-terminal or the C-terminal residue of the PNA. This peptide is a substrate for protein kinase A, which phos-phorylates the critical Ser residue. This phosphorylation was carried out using [y-32P]ATP. Kozlov et al. 45 made 2 -deoxycytidine-3 -phosphoroimidazolide to react with the N-terminus of a PNA in solution. T4 polynucleotide kinase then phosphorylated the 5 -hydroxy group of the nucleotide by [y-32P]ATP. 

Weinfeld M, Liuzzi M, Paterson MC (1990) Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites design of a 32P-postlabeling assay for apurinic sites in DNA. Biochemistry 29 1737-1743... 

The pEU3b plasmid was digested with Spe I and blunted with T4 polynucleotide kinase. The plasmid was then digested with Sal I. The coding region for yeast ubiquitin was prepared as follows. 

P-Radiolabeled RNA substrate to be cleaved (prepared by reaction of the RNA with y-32P-ATP and T4 polynucleotide kinase alternatively, 3/-32P-radiolabeling with 32P-pCp and T4 RNA ligase can be used)... 

T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) phenol-chloroform (Gibco BRL, Rockville, MD, USA) 732P-AIP (NEN, Boston, MA, USA) Chroma Spin-10 columns (Clontech, Palo Alto, CA, USA) ExpressHyb Hybridization Solution (Clontech, Palo Alto, CA, USA) 20 x saline-sodium citrate (SSC) and 10% sodium dodecyl sulfate (SDS Biofluids, Rockville, MD). 

Oligonucleotide primers are synthesized with an Applied Biosystems 380A synthesizer by the phosphoramidite method T4 polynucleotide kinase (10 U//il) is obtained from Amersham (Amer-sham, UK)... 

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

The protocol for EMSA as recently noted by Xu et al. 2004 is as follows. Oligonucleotides are labeled using T4 polynucleotide kinase and [y -32P] ATP. The labeled probes are purified from free nucleotides by ethanol precipitation. 5 pL of extracted proteins is mixed with 2 xL of 5 x gel shift binding buffer and incubating at room temp for 15 min. Then 1 pL of 32P-labeled oligonucleotide is added and 20 min is given for a binding reaction to occur at room temp. 

There are two reports describing the use of nucleosides with other sugar conformations. A method for preparing internally P-labelled l-DNA has been described using T4 polynucleotide kinase. Chimeric DNA composed of tandem a- and p-anomeric strands have been used in TFOs and shown to have enhanced thermal stability compared to all a- or p-anomeric oligonucleotides. ... 

Q.Convert the insert io the DNA probe At this point, the test tube contains a large quanlitj of the insert. The goal is to convert the insert to a form usable as a probe. T4 polynucleotide kinase is used to catalyze the attachment of radioactive phosphate to the ends of the DMA ... 

An alternative method to prepare correct termini for the ligation with T7 RNA ligase is to dephosphorylate both ends of the 5 -fragment with E. coli alkaline phosphatase and phosphorylate both ends of the 3 -fragment with T4 polynucleotide kinase.71 72... 

T4 polynucleotide kinase (T4 PNKase) transfers the terminal phosphate (i.e., gamma position) of ATP to 5 -OH termini of DNA or... 

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