Molecular Cloning: A Laboratory Manual, Book 2 |
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Page 8-19
... HindIII Pvull Hindill RNAase H bacteriophage T4 polynucleotide kinase FIGURE 8.6 Cloning cDNA by the Okayama - Berg method . Cleave the double - stranded cDNA with the appropriate restriction. E. coli DNA polymerase I E. coli DNA ligase ...
... HindIII Pvull Hindill RNAase H bacteriophage T4 polynucleotide kinase FIGURE 8.6 Cloning cDNA by the Okayama - Berg method . Cleave the double - stranded cDNA with the appropriate restriction. E. coli DNA polymerase I E. coli DNA ligase ...
Page 8-39
... ( HindIII / BamHI / EcoRI ) at codons 5-10 of the lacZ gene ( see map of λORF8 in Chapter 2 , page 2.50 ) . Insertion ... HindIII . Because M.HindIII methylase is not commercially available , the cDNA is methylated with M.BamHI methylase ...
... ( HindIII / BamHI / EcoRI ) at codons 5-10 of the lacZ gene ( see map of λORF8 in Chapter 2 , page 2.50 ) . Insertion ... HindIII . Because M.HindIII methylase is not commercially available , the cDNA is methylated with M.BamHI methylase ...
Page 8-41
... HindIII site is formed by fusion of the poly ( dT : dA ) tract at the 3 ' terminus of the cDNA to the first linker ( see Figure 8.13 ) . Addition of linkers to the 5 ' terminus of the cDNA does not usually create a new restriction site ...
... HindIII site is formed by fusion of the poly ( dT : dA ) tract at the 3 ' terminus of the cDNA to the first linker ( see Figure 8.13 ) . Addition of linkers to the 5 ' terminus of the cDNA does not usually create a new restriction site ...
Contents
E | 8-8 |
SYNTHESIS OF THE FIRST STRAND OF CDNA 8 | 8-11 |
CONCENTRATING NUCLEIC ACIDS BY EXTRACTION WITH BUTANOL E | 8-16 |
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acrylamide agarose gel aliquots amplification annealing antibody bacteriophage M13 bacteriophage T4 bacteriophage T4 DNA buffer carried cDNA clones cDNA libraries cells centrifugation at 12,000g cleavage cleaved cleotide coli DNA polymerase concentration containing ddNTPs deletions denatured digestion diluted dithiothreitol DNA ligase DNA sequencing DNAase dNTPs double-stranded DNA EDTA EDTA pH 8.0 ethanol exonuclease expression libraries formamide fusion proteins gel electrophoresis gene genomic DNA H₂O hybridization Klenow fragment labeled ligation linear linkers Maxam-Gilbert method MgCl2 microfuge tube minutes at 4°C molecules mRNA Natl nitrocellulose nitrocellulose filters Nucleic Acids nucleotides oligonucleotide phagemid piperidine plaques plasmid plasmid DNA plate polymerase chain reaction precipitation prepared primer probe purified radioactivity radiolabeled recombinant remove restriction enzyme room temperature samples Sequenase sequencing gel sequencing reactions sonicated specific activity stored strand of cDNA synthesis T4 DNA polymerase target DNA target sequence template DNA termini transcription transfer Tris Cl pH vectors µg/ml