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A precaution when preparing very large plasmids by alkaline lysis procedure
N R Thomas1, S Koshy, M Simsek
1Department of Biochemistry, Faculty of Medicine, Kuwait University.
Biotechnology and Applied Biochemistry
|October 1, 1988
Summary
Potassium acetate buffer selectively removes large plasmids during DNA extraction using sodium lauryl sulfate (SDS). Using N-lauryl sarcosine detergent prevents this loss, suggesting faster precipitation traps large plasmids.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Plasmid DNA extraction is crucial for molecular biology techniques.
- The sodium lauryl sulfate (SDS)-alkali method is a common DNA extraction procedure.
- Neutralization conditions can impact DNA recovery, particularly for large plasmids.
Purpose of the Study:
- To compare the extractability of plasmids of varying sizes using the SDS-alkali method.
- To investigate the effect of different neutralizing agents (sodium acetate vs. potassium acetate) on plasmid recovery.
- To evaluate the role of detergent type in large plasmid loss.
Main Methods:
- Comparison of plasmid extractability using SDS-alkali lysis with sodium acetate or potassium acetate buffers.
- Assessment of large plasmid ( > 100 kb) recovery under different buffer conditions.
- Evaluation of N-lauryl sarcosine as an alternative detergent to SDS.
- Kinetic analysis of precipitate formation with sodium acetate and potassium acetate.
Main Results:
- Selective loss of large plasmids (> 100 kb) was observed when using potassium acetate buffer with SDS.
- No significant loss of large plasmids occurred when N-lauryl sarcosine was used as the detergent, even with potassium acetate.
- Precipitation of lauryl sulfate was slower and less extensive with sodium acetate compared to potassium acetate.
- Faster precipitation kinetics with potassium acetate are hypothesized to trap large, denatured plasmids.
Conclusions:
- The choice of neutralizing agent significantly impacts the recovery of large plasmids during SDS-alkali DNA extraction.
- Potassium acetate promotes a faster precipitation that can lead to the entrapment of large plasmids.
- N-lauryl sarcosine offers a viable alternative detergent for preserving large plasmid integrity during extraction, especially in the presence of potassium ions.