A precaution when preparing very large plasmids by alkaline lysis procedure.
The extractabilities of plasmids of different sizes by the sodium lauryl sulfate (SDS)-alkali procedure were compared using either sodium acetate or potassium acetate buffer as the neutralizing agent. There was a selective loss of large plasmids (above 100 kb) when the potassium salt was used. When N-lauryl sarcosine instead of SDS was used as the detergent, no loss of large plasmids occurred in the presence of potassium salt. A comparison of the kinetics of precipitate formation with sodium acetate and potassium acetate indicated that the rate and the amount of lauryl sulfate precipitated were lower with the sodium salt. It is suggested that faster precipitation of lauryl sulfate with potassium acetate leads to trapping of large denatured plasmids that cannot renature as fast as the small ones.
Thomas
N R
NR
Department of Biochemistry, Faculty of Medicine, Kuwait University.
Koshy
S
S
Simsek
M
M
Abraham
A K
AK
D000085:Acetates; D019342:Acetic Acid; D000468:Alkalies; D002499:Centrifugation, Density Gradient; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D007700:Kinetics; D010957:Plasmids; D012967:Sodium Dodecyl Sulfate
D000085:Acetates; D000468:Alkalies; D012967:Sodium Dodecyl Sulfate; D004262:DNA Restriction Enzymes; D004996:Ethidium; D019342:Acetic Acid