Molecular Cloning: A Laboratory Manual, Book 2 |
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Page 15-10
... reaction for 6-8 hours at 16 ° C . 10x Blunt - end ligation buffer · 0.66 M Tris Cl ( pH 7.6 ) 50 mM MgCl2 50 mm dithiothreitol 1 mg / ml bovine serum albumin ( Fraction V ; Sigma ) ( optional ) 10 mm hexamminecobalt chloride 2 mM ATP 5 ...
... reaction for 6-8 hours at 16 ° C . 10x Blunt - end ligation buffer · 0.66 M Tris Cl ( pH 7.6 ) 50 mM MgCl2 50 mm dithiothreitol 1 mg / ml bovine serum albumin ( Fraction V ; Sigma ) ( optional ) 10 mm hexamminecobalt chloride 2 mM ATP 5 ...
Page 15-30
... ligation reaction should not exceed 5-6 nM . This concentration is equivalent to 10 μg / ml of a plasmid of the size of pBR322 . If desired , a phosphorylated linker can be inserted at the site of recircularization by including the ...
... ligation reaction should not exceed 5-6 nM . This concentration is equivalent to 10 μg / ml of a plasmid of the size of pBR322 . If desired , a phosphorylated linker can be inserted at the site of recircularization by including the ...
Page 15-44
... reaction . For example , dissolve 500 ng of linearized plasmid DNA and 100 ... ligation mixtures at -20 ° C until step 2 of the protocol has been completed ... ligation reaction ( s ) yielded the largest number ( s ) of Kan ' Amp ...
... reaction . For example , dissolve 500 ng of linearized plasmid DNA and 100 ... ligation mixtures at -20 ° C until step 2 of the protocol has been completed ... ligation reaction ( s ) yielded the largest number ( s ) of Kan ' Amp ...
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