DNA extraction from 0.22 microM Sterivex filters and cesium chloride density gradient centrifugation.
This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 microM Sterivex filters that have been treated with storage/lysis buffer and archived at -80 degrees C, and to purify this DNA using a cesium chloride density gradient. The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA. Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract. Part Five describes the optional purification via cesium chloride density gradient. It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.
Wright
Jody J
JJ
Department of Microbiology and Immunology, University of British Columbia.
Lee
Sangwon
S
Zaikova
Elena
E
Walsh
David A
DA
Hallam
Steven J
SJ
D002499:Centrifugation, Density Gradient / Q000379:methods*; D002586:Cesium / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry*; D004247:DNA / Q000302:isolation & purification*; D005374:Filtration / Q000295:instrumentation* / Q000379:methods; D010933:Plankton / Q000737:chemistry / Q000235:genetics
D002712:Chlorides; D002586:Cesium; D004247:DNA; C028019:cesium chloride