04 U/[micro]L GlaI endonuclease, and 33 U/[micro]l T4 DNA ligase
Chen, "Ischemic preconditioning induces XRCC1, DNA polymerase-beta, and DNA ligase
III and correlates with enhanced base excision repair," DNA Repair, vol.
Finally, DNA ligase
catalyses the formation of two new phosphodiester bonds and thus seals the sugar-phosphate backbone.
Annealed Y adaptors (5'-(PO4-) GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3' and 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') were ligated by using T4 DNA Ligase
in Rapid Ligation Buffer (Enzymatics, Beverly, MA, USA).
Repair of oxidative and alkylation DNA damage in mitochondria through BER occurs in a manner similar to that of nDNA with several modifications: Gap filling in both short- and long-patch repair is carried out by polymerase gamma and its accessory subunits, DNA ligase
III and EXOG (which carries out 5' to 3' exonuclease activity in long-patch BER) (Cymerman et al.
1) In NHEJ in vertebrate cells, seven molecules playing pivotal role have been identified: Ku70, Ku86 (also known as Ku80), DNA-PKcs (DNAdependent protein kinase catalytic subunit), Artemis, XRCC4, DNA ligase
IV (Lig4) and XRCC4-like factor (XLF, also known as Cernunnos).
An appropriate amount of prepared plasmid (pUB110) and insert were mixed for ligation reaction with T4 DNA ligase
O processo e simples e baseia-se em dois tipos de enzimas, as enzimas de restricao e a enzima DNA ligase
coli DH5 (Alpah) and Rosseta host strains were obtained commercially; pMD18-T vector, DNA Marker, Rnase A, isopropyl- (Beta) -D-thiogalactopyranoside (IPTG), TaKaRa Ex Taq, T4 DNA Ligase
, 5-bromo-4-chloro-3-lndolyl- (Beta) -D- galactoside (X-gal), EcoR I, Not I and gel extraction purification kit were from TaKaRa (Dalian, Japan); Tryptone and yeast extract were from OXOID (England); Acrylamide (Acr), N,N'-methylenebisacrylamide (Bis), sodium dodecyl sulfate (SDS) and tetramethylethylenedia -mine (TEMED) were from Sigma (USA); All other chemicals were analytical grade from commercial sources; All primers were synthesized by Sangon Biotech (Shanghai, China).
The digested DNA was ligated with T4 DNA ligase
to a final volume of 7.
Next, 10[micro]L of a solution containing 1[micro]g of EcoRI and Msel adapters, 1X T4 DNA ligase
buffer and 1U of T4 DNA ligase
The main reagents used in the present study were AxyPrep-96 whole blood genomic DNA (AXYGEN, USA), Taq DNA polymerase (Qiagen Hotstar, Germany), Taq DNA ligase
enzyme (NEB, UK), and dNTP (Promega, USA).
The kit combines TurboLigase[TM], an ultrapure recombinant T4 DNA Ligase
, and 5x TurboLigation buffer, an optimized quick ligation buffer.
The amplified gene of cellulase was purified from the agarose gel after electrophoresis using Qiagen gel extraction kit and ligated into the pTZ57R/T cloning vector (Fermentas) using DNA ligase
and ligation buffer.
Descriptively, the hOGG1 gene on chromosome 3p26 catalyzes the cleavage of a glycosylic bond between the modified base and a sugar moiety, leaving an abasic apurinic/apyrimidinic site in DNA which is then incised followed by a few successive actions of a phosphodiesterase that completes the repair process with a final function by a DNA polymerase, and a DNA ligase