T4 DNA ligase

T4 DNA ligase

Product description:

English name: T4 DNA Ligase
CAS: 9015-85-4

Molecular Weight: 55.3kDa, monomer

Level: BR
Source: T4 phage clone containing the E. coli gene 30
Concentration: 5Weiss u / ul (1000CEU / ul)
Activity Definition: refers to the ATP-PPi exchange reaction, 37 ℃, within 30 minutes 1nmol [32PPi] converted to Norit can absorb the amount of enzyme required to form (weiss unit, a unit equivalent to approximately 200 weiss sticky ends a connecting unit connecting unit sticky ends:. 20ul reaction system (50mM Tris-HCL (PH7.5), 10mM DTT, 10mM MgCL2, 1mM ATP, 25ug / ml BSA, 12uM (300ug / ml) of 5-DNA terminus) In at 16 ℃, 50% connection to connect the amount of enzyme HindIII digested Lambda DNA desired product within 30 minutes)
Activity assay mixture: 66mM Tris-HCL (PH7.6), 10mM DTT, 6.6mM MgCL2, 0.066mM ATP, 3.3uM [32PPi]
Preservation solution components: 20mM Tris-HCL (PH7.5), 50mM KC1, 1mM DTT, 0.1mM EDTA and 50% (v / v) glycerol
10 * T4 DNA Ligase Buffer (# B69): 400mM Tris-HCL, 100mM MgCL2, 100mM DTT, 5mM ATP (PH7.525 ℃)
Inhibitors: When the reaction system NaC1 or KC1 concentration exceeds 200 mM, can strongly inhibit the activity of T4 DNA Ligase
Inactivation: 65 ℃ heated for 10 minutes or 70 ℃ heated 5 minutes
Note: T4 DNA Ligase and DNA agarose gel electrophoresis in combination will change the mobility of bands, in order to avoid this phenomenon, the sample to be treated prior to electrophoresis or molecular weight standard sample processing as follows: Samples with Fermentas Company 6 * DNA Loading Dye & SDS Solution ( # R1151) mixing, 75 ℃ heated for 5 minutes or 65 ℃ was heated for 10 minutes the ice bath was saved; the conversion, the volume of the ligation product of the volume of competent cells should not exceed 10%; Before electroporation, the first removed by chloroform extraction method The ligation mixture T4 DNA Ligase, and then extract the product was purified by ethanol precipitation
Quality control: tests show no endodeoxyribonucleases, RNase, phosphatase pollution function test: DNA sticky ends or blunt ends connectivity
Characters: liquid formed between the enzyme catalyzes double-stranded DNA or RNA adjacent 5 'phosphate groups and 3'-hydroxyl termini phosphodiester bond, can also repair double-stranded DNA, RNA or DNA / RNA complex in single strand incision, connecting DNA sticky peaceful ends, but the single-stranded nucleic acid enzyme no activity. The enzyme needs cofactors ATP
Use: Biochemical studies connecting sticky ends or blunt double-stranded DNA; and double-stranded and double-stranded DNA oligonucleotide linker connection; double-stranded DNA, RNA or DNA-RNA complex in the nick of repair; ligase mediated. RNA testing; site-directed mutagenesis; amplified fragment length polymorphism
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