Topic
Lysis buffer
About: Lysis buffer is a research topic. Over the lifetime, 763 publications have been published within this topic receiving 11594 citations.
Papers published on a yearly basis
Papers
1 Jan 1997
TL;DR: The method described below, which is a modification of a procedure described by Longmfreet al. (1991), has several advantages: relatively small amounts of tissue routinely provide large quantities of DNA, including both nuclear and mitochondrial components.
Abstract: Many molecular techniques requfre relatively large quantities of high molecular weight DNA and several protocols for collection are well established if there are unlimited niunbers of samples or if all procedures can be performed tmder the stable conditions afforded in a laboratory. Unfortunately, the coUection of DNA for many species, populations, or samples does not afford such stable conditions. Conservation genetic studies of rare and endangered species often force opportunistic coUections which involve road or other accidental kills, exploitation of dead animals at zoos, collection under exfreme field conditions, etc. The method described below, which is a modification of a procedure described byLongmfreet al. (1991), has several advantages. First, relatively small amounts of tissue routinely provide large quantities of DNA, including both nuclear and mitochondrial components. Second, this method consistently yields high molecular weight DNA that can be used for all molecular techiuques including cosmid library constinction. Thfrd, nuclear DNA can be obtained from essentiaUy any tissue that contains nucleated cells. This would include, but not be limited to, nucleated red blood cells of lower vertebrates and bfrds, muscle, heart, liver, kidney, testes, embryonic tissue from placentas or eggs. pulp of growing feathers, skin biopsies, and hafr follicles. Fourth, samples coUected in lysis buffer do not requfre refrigeration. Fifth, field procedures are relatively simple and inexpensive. Sixth, scissors and forceps are the only eqmpment that need to be cleaned between samples to protect against cross contamination. Finally, a small kit and limited equipment allows one to be prepared for opportunistic situations.
497 citations
TL;DR: An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed that circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.
Abstract: Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC. An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.
361 citations
TL;DR: A simple and fast, one-step protocol to remove impurities highly efficiently from E. coli proteins, endotoxins, and detergents is described and it is shown that proteins are able to refold correctly after isopropanol treatment.
260 citations
TL;DR: A microfluidic lysis device is developed that can achieve complete lysis of erythrocytes and approximately 100% recovery of leukocytes where the cells are exposed to an isotonic lysis buffer for less than 40 s, after which theLeukocytes are immediately returned to physiological conditions.
Abstract: Leukocyte isolation from whole blood to study inflammation requires the removal of contaminating erythrocytes. Leukocytes, however, are sensitive to prolonged exposure to hyper/hypoosmotic solutions, temperature changes, mechanical manipulation, and gradient centrifugation. Even though care is taken to minimize leukocyte activation and cell loss during erythrocyte lysis, it is often not possible to completely avoid it. Most procedures for removal of contaminating erythrocytes from leukocyte preparations are designed for bulk processing of blood, where the sample is manipulated for longer periods of time than necessary at the single-cell level. Ammonium chloride-mediated lysis is the most commonly used method to obtain enriched leukocyte populations but has been shown to cause some activation and selective loss of certain cell types. The leukocyte yield and subsequent activation status of residual leukocytes after NH4Cl-mediated lysis have been shown to depend on the time of exposure to the lysis buffer. W...
175 citations
TL;DR: Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two‐dimensional (2‐D) polypeptide patterns and major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins.
Abstract: Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with “standard” lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with “standard” lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea / 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of “standard” lysis buffer (procedure iii).
174 citations
Related Topics (5)
Performance Metrics
| Year | Papers |
|---|---|
| 2025 | 3 |
| 2024 | 12 |
| 2023 | 35 |
| 2022 | 32 |
| 2021 | 25 |
| 2020 | 43 |