Molecular Cloning: A Laboratory Manual, Book 3 |
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Page 60
Centrifuge the suspension of disrupted cells at 12 , 000g for 5 minutes at 4°C in a
microfuge . Transfer the supernatant to a fresh microfuge tube . Reserve 50 ul for
the CAT assay , and store the remainder of the extract at - 20°C . 4 . Incubate ...
Centrifuge the suspension of disrupted cells at 12 , 000g for 5 minutes at 4°C in a
microfuge . Transfer the supernatant to a fresh microfuge tube . Reserve 50 ul for
the CAT assay , and store the remainder of the extract at - 20°C . 4 . Incubate ...
Page 19
Heat the reaction for 5 minutes at 68°C to inactivate the bacteriophage T4
polynucleotide kinase . Place on dry ice and store at - 20°C . 3 . Hybridize the
primer to template bacteriophage M13 single - stranded DNA carrying the cloned
gene ...
Heat the reaction for 5 minutes at 68°C to inactivate the bacteriophage T4
polynucleotide kinase . Place on dry ice and store at - 20°C . 3 . Hybridize the
primer to template bacteriophage M13 single - stranded DNA carrying the cloned
gene ...
Page 37
Incubate the suspension at 30°C . After 15 minutes , take out two small aliquots of
the suspension . To one sample , add Nonidet P - 40 ( NP - 40 ) to a final
concentration of 1 % . Examine the two samples by phase - contrast microscopy .
Incubate the suspension at 30°C . After 15 minutes , take out two small aliquots of
the suspension . To one sample , add Nonidet P - 40 ( NP - 40 ) to a final
concentration of 1 % . Examine the two samples by phase - contrast microscopy .
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Contents
BOOK | 3 |
Identification and Analysis of Recombinants 2 108 | 30 |
TRANSFECTION MEDIATED BY DEAEDEXTRAN 16 | 41 |
Copyright | |
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Common terms and phrases
acetate activity added Adjust aliquots allow amount antibody antigen Appendix appropriate approximately aqueous assay bacterial bacteriophage base binding buffer carried cells centrifugation Chapter chromatography Cl pH cloned coli column complete concentration containing culture deionized water described desired detected dilution Dissolve EDTA efficiency electrophoresis enzyme et al ethanol expression extract Figure filter final fragment gene growth hybridization Incubate levels ligation mammalian cells medium method mg/ml microfuge tube minutes mixture mRNAs nucleic acid pellet phase phosphate Place plasmid plate polymerase precipitation prepared procedure Products promoter protein radioactive radiolabeled reaction remove restriction room temperature sample screen selection sequence sodium solution specific step sterile stored strain TABLE Telephone termini tion transcription transfection transfer transformation Tris ug/ml usually vectors volume wash western blotting ОН