Patent ID: 7754873

Claim:
A method for isolating plasmid DNA from bacteria via alkaline lysis, the method comprising: (a) suspending bacterial cells in a P 1 buffer with a pH of between 7-9; wherein the P 1 buffer contains a dye for monitoring suspension of the bacterial cells, whereby adding the P 1 buffer results in a colored bacterial suspension allowing visual inspection of the colored bacterial suspension ensuring efficient suspension of the bacteria from added contrast resulting from the colored bacterial suspension; (b) adding a sufficient amount of P 2 buffer with a basic pH of between 11-14 to the bacterial suspension, wherein the P 2 buffer contains a dye for monitoring the lysis of the bacterial suspension, whereby adding and mixing the P 2 buffer to the bacterial suspension results in efficient lysis producing a bacterial lysate and wherein the color of the lysate changes and clarifies indicating that lysis of the bacterial suspension is complete and that the P 2 buffer was effective in causing efficient lysis; (c) precipitating cellular debris by adding a sufficient amount of P 3 neutralization buffer with a pH of between 3-6 producing a cleared bacterial lysate containing increased amounts plasmid DNA due to the efficient neutralization; wherein the P 3 buffer contains a dye; and wherein the color of the cleared bacterial lysate changes upon adding the P 3 buffer indicating the P 3 buffer was effective in causing efficient neutralization; (d) removing cellular debris from the cleared bacterial lysate by filtering or centrifuging to obtain a lysate filtrate, and then adsorbing the plasmid DNA in the lysate by passing the lysate filtrate over a DNA binding matrix, whereby the dye or dyes in the colored cleared lysate passes or pass through the DNA binding matrix and thereby is or are discarded as part of the waste flow through; (e) washing the adsorbed plasmid DNA on the DNA binding matrix by adding wash buffer and filtering or centrifuging to obtain a clear flow through of wash buffer, discarding the clear flow through wash buffer; and (f) eluting the plasmid DNA from the DNA binding matrix by adding an elution buffer and filtering or centrifuging to obtain a plasmid DNA containing eluate.