English name: T4 DNA Ligase
CAS: 9015-85-4
Molecular Weight: 55.3kDa, monomer
Source: T4 phage clone containing the E. coli gene 30
Concentration: 1u / ul (200CEU / 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 ℃)
50% PEG solution: 50% (w / v) polyethylene glycol 4000
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: polyethylene glycol (PEG) can significantly increase the efficiency of the connection of blunt-ended DNA, PEG4000 recommended concentration in the reaction system in an amount of 5% (w / v); T4 DNA Ligase and DNA binding strip will change agarose electrophoretic mobility, in order to avoid this phenomenon, prior to electrophoresis treatable sample or molecular weight standard sample processing as follows: Samples with Fermentas Company 6 * DNA Loading Dye & SDS Solution (# R1151) mixing, 75 ℃ heat 5 minutes or 65 ℃ heating 10 After 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, first remove the ligation mixture using T4 DNA Ligase chloroform extraction method, and then extracted and purified by ethanol precipitation product
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
Usage: research, experiments sticky ends or blunt ends connected double-stranded DNA; double-stranded and double-stranded DNA oligonucleotide linker is connected; double-stranded DNA, RNA, or DNA-RNA complex in the gap repair; ligase-mediated The RNA detection; site-directed mutagenesis; amplified fragment length polymorphism
Storage conditions: -20 ° C storage