Molecular cloning: a laboratory manual, Volume 1 |
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Page 1-25
Incubate the culture overnight at 37°C with vigorous shaking. 2. Pour 1.5 ml of the
culture into a microfuge tube. Centrifuge at 12,000g for 30 seconds at 4°C in a
microfuge. Store the remainder of the culture at 4°C. 3. Remove the medium by
aspiration, leaving the bacterial pellet as dry as possible. The supernatant can be
conveniently removed with a disposable pipette tip attached to a vacuum line (
see Figure 1.3). Use a gentle vacuum and touch the tip to the surface of the liquid
.
Incubate the culture overnight at 37°C with vigorous shaking. 2. Pour 1.5 ml of the
culture into a microfuge tube. Centrifuge at 12,000g for 30 seconds at 4°C in a
microfuge. Store the remainder of the culture at 4°C. 3. Remove the medium by
aspiration, leaving the bacterial pellet as dry as possible. The supernatant can be
conveniently removed with a disposable pipette tip attached to a vacuum line (
see Figure 1.3). Use a gentle vacuum and touch the tip to the surface of the liquid
.
Page 7-7
Collect the cells by centrifugation at 2000g for 5 minutes at 4°C. Wash the cell
pellet three times by resuspension in 10 volumes of ice-cold PBS lacking calcium
and magnesium ions; use a wide- bore pipette to disperse the cell pellet gently,
but completely, each time. b. Estimate the volume of the packed cells, and
resuspend them in 10-20 volumes of RNA extraction buffer. c. Add a volume of
proteinase digestion buffer equal to the volume of RNA extraction buffer added in
b. Mix the ...
Collect the cells by centrifugation at 2000g for 5 minutes at 4°C. Wash the cell
pellet three times by resuspension in 10 volumes of ice-cold PBS lacking calcium
and magnesium ions; use a wide- bore pipette to disperse the cell pellet gently,
but completely, each time. b. Estimate the volume of the packed cells, and
resuspend them in 10-20 volumes of RNA extraction buffer. c. Add a volume of
proteinase digestion buffer equal to the volume of RNA extraction buffer added in
b. Mix the ...
Page 7-62
Store the solution at 0°C for at least 30 minutes. Recover the nucleic acids by
centrifuga- tion at 12,000g for 15 minutes at 4°C in a microfuge. Discard the
supernatant, wash the pellet with 70% ethanol, and recentrifuge. Carefully
remove all of the ethanol, and store the pellet of nucleic acids at room
temperature until the last visible traces of ethanol have evaporated. Do not allow
the pellet to become desiccated, otherwise it will be difficult to dissolve. 3.
Redissolve the pellet of nucleic ...
Store the solution at 0°C for at least 30 minutes. Recover the nucleic acids by
centrifuga- tion at 12,000g for 15 minutes at 4°C in a microfuge. Discard the
supernatant, wash the pellet with 70% ethanol, and recentrifuge. Carefully
remove all of the ethanol, and store the pellet of nucleic acids at room
temperature until the last visible traces of ethanol have evaporated. Do not allow
the pellet to become desiccated, otherwise it will be difficult to dissolve. 3.
Redissolve the pellet of nucleic ...
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LibraryThing Review
User Review - iayork - LibraryThingthe BIBLE of every biologist: So few and so much to say about this bible of Biology at the bench... You'll really find everything you want in it, including the composition of all the buffers and ... Read full review
Contents
Replication and Incompatibility 1 | 1-3 |
Constructing Expression Libraries in Plasmid and Bacteriophage | 1-12 |
Oligonucleotidemediated Mutagenesis 15 51 | 1-15 |
Copyright | |
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Common terms and phrases
agar plate agarose gel aliquots ampicillin antibiotic bacteriophage particles bacteriophage T4 DNA BamHI buffer carry cDNA cells centrifugation at 12,000g Cl pH cleaved cloning coli containing cosmid cosmid vector culture digestion DNA fragments DNA ligase DNA molecules DNA polymerase double-stranded DNA EcoRI EDTA EDTA pH 8.0 efficiency Escherichia coli ethanol ethidium bromide eukaryotic exonuclease extracts final concentration foreign DNA formamide gel electrophoresis gene genetic gradients Hindlll host hours at 37°C hybridization Incubate infected inserted ligation reaction linear lysogenic method microfuge tube minutes at 4°C minutes at room mixture mutations nitrocellulose nitrocellulose filter Nucleic Acids nucleotides origin of replication packaging pellet phagemid plaques plasmid DNA plasmid vectors polycloning prepared probes protein purified recA recombinant remove replication restriction enzymes RNAase room temperature segment of foreign sequences single-stranded DNA solution step sterile stored strains strand supernatant T4 DNA ligase teriophage transfer transformation vector DNA vitro volume
References to this book
Bibliographic information
| Title | Molecular cloning: a laboratory manual, Volume 1 Molecular Cloning: A Laboratory Manual, E. F. Fritsch, ISBN 0879693096, 9780879693091 |
| Authors | Joseph Sambrook, E. F. Fritsch, Tom Maniatis |
| Photographs by | E. F. Fritsch |
| Edition | 2, illustrated |
| Publisher | Cold Spring Harbor Laboratory, 1989 |
| Original from | the University of Michigan |
| Digitized | 23 Jul 2008 |
| ISBN | 0879693096, 9780879693091 |
| Length | 3 pages |
| Subjects | › › › Eukaryotic cells Medical / Genetics Molecular cloning Science / Life Sciences / Biology / Molecular Biology Science / Life Sciences / Genetics & Genomics |
| Export Citation | BiBTeX EndNote RefMan |