Potassium Chloride → Phenol chloroform

Participants

Reaction Steps

To further examine the ability of Topo65 to mediate ligation of DNAs containing 5′-briding phosphorothiolate near the 5′ end of the scissile strand, a vector was adapted with duplex oligonucleotides TCRIBamS/AS described in Example II. FIG. 6A depicts the linearized and oligonucleotide adapted vectors. T4 DNA ligase was then used to adapt the vector ends with TCRIBamS oligonucleotide and TCRIBamAS oligonucleotides that contained or lacked a 5′ phosphate. EcoRI-digested vector was incubated at 16° C. overnight with a 300-fold molar excess of oligonucleotides. The adapted vector was then incubated in the presence or absence of Topo65 in 10-fold molar excess, overnight at room temperature in a reaction buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, 1 mM EDTA and 2 mM DTT. After this incubation, 5 M NaCl was added to obtain a final concentration of 600 mM, and the mixture remained at room temperature for three hours. To this mixture, SDS was added to a final concentration of 0.1%, Proteinase-K was added to a final concentration of 100 μg/ml, and the mixture was incubated at 37° C. for 30 minutes. The DNA was isolated by phenol/chloroform extraction, followed by ethanol precipitation. The isolated DNA was used to transform TOP10F′ cells, which were then plated onto 2YT/Ampicillin agar.

Provenance & Evidence