Lab #8 info

Lab #8 info - Laboratory: VIII Topic: Quantitation of DNA...

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Laboratory: VIII Topic : Quantitation of DNA by UV spectrophotometry and Plasmid restriction digest Introduction: Quantitation of DNA by UV spectrophotometry : Crude DNA preparations often contain proteins, RNA as well as ethanol and salts associated with the DNA isolation procedures. The purity and concentration of a DNA sample can be analyzed using spectrophotometry. DNA purity is estimated using the ratio of absorbances (also known as optical density (OD) at 260 and 280 nm. The A 260 /A 280 value should fall in the range of 1.8 – 2 for good quality DNA. The concentration of a DNA sample is determined by measuring the absorbance at 260nm. A pure DNA solution in distilled water with an absorbance of 1 at 260nm has a concentration of 50 μ g/ ml of DNA. The absorbance is used to calculate the concentration of unknown DNA samples. Crude DNA preps extracted without RNAse enzyme contain with RNA. This may skew your DNA concentration calculation. DNA agarose gel analysis (next lab) will confirm the absence/ presence of RNA in your isolated plasmid. Restriction Enzymes : Viruses called bacteriophages are major enemies of bacteria. These viruses infect bacteria by injecting their own DNA into bacteria to force the bacteria to multiply the DNA. Bacteria have responded by evolving a natural defense, called restriction enzymes, to cut up and destroy the invading DNA; Restriction enzymes search the DNA for specific palindromic sequences of base pairs, such as GAATTC , and cut the DNA at these sites. The actual sequence of DNA is called a restriction site . . Restriction sites are palindromes (read same on upper and lower strand in the 5’ to 3’ direction) Examples: EcoR 1 - 5’ GAATTC 3’ Hind III - 5’ AAGCTT 3’ top strand 3’ CTTAAG 5’ 3’ TTCGAA 5’ bottom strand The restriction enzymes are named according to the bacterial strain from which the enzyme was first isolated. EcoR 1 was the first enzyme isolated from E scherichia co li R Y13 strain. Hind III was the third enzyme isolated from H aemophilus i nfluenzae Rd strain. Several computer software programs and websites are available to draw restriction maps and to determine the size of the fragments formed after digestion. Restriction maps show the different restriction enzyme sites present in a DNA sequence. Restriction enzymes are important DNA analysis and in recombinant technology. DNA restriction fragments can be cloned into DNA vectors (example: plasmids) to prepare recombinant DNA molecules used for protein synthesis and in gene analysis. pGLO plasmid
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This note was uploaded on 03/09/2011 for the course PHS 2301 taught by Professor Bohu during the Spring '11 term at St. John's.

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Lab #8 info - Laboratory: VIII Topic: Quantitation of DNA...

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