IUB BIOL-L 211 - PCR, DNA Sequencing, and Restriction Endonucleases (3 pages)

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PCR, DNA Sequencing, and Restriction Endonucleases



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PCR, DNA Sequencing, and Restriction Endonucleases

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Polymerase Chain Reaction and DNA Sequencing and Restriction Endonucleases


Lecture number:
10
Pages:
3
Type:
Lecture Note
School:
Indiana University, Bloomington
Course:
Biol-L 211 - Molecular Biology
Edition:
1
Documents in this Packet

Unformatted text preview:

BIOL L 211 Lecture 10 Outline of Last Lecture I DNA Polymerase II Sliding Clamp Loader III Finishing Replication IV E Coli Holoenzyme and Replisome Outline of Current Lecture I Polymerase Chain Reaction II Agarose Gel Electrophoresis III Restriction Endonucleases Current Lecture Polymerase Chain Reaction and DNA Sequencing and Restriction Endonucleases I Polymerase Chain Reaction PCR A Definition one of the most widely used laboratory techniques used to copy genes using a method similar but not identical to natural DNA replication 1 Note Performed outside of cell i e in a lab B PCR Cycle the completion of the three steps below each cycle doubles amount of DNA 1 Denaturing hydrogen bonds between base pairs are broken via heating all at once yielding no replication forks and two separate single DNA strands 2 Annealing DNA primers NOT RNA are added to both DNA strands with no 3 Extension DNA polymerase recognizes the primer template junction and extends the sequence in the 5 to 3 direction in one continuous stretch leading lagging strands C Ingredients These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute 1 DNA template can be genomic or from plasmid 2 Set of DNA primers synthetic in order to emphasize certain regions 3 Modified DNA polymerase can tolerate high heat commits fewer errors and proceeds without the help of a sliding clamp 4 Solution with nucleotides and appropriate salt mixture D Note be able to compare contrast PCR with natural DNA Replication II Agarose Gel Electrophoresis A Definition process that separates DNA fragments by relative size using electrical charge B Procedure 1 Agarose gel produced from compounds including ethidium bromide 2 Chamber prepared with gel and salt buffer 3 Combined DNA and heavy dye are put into well 4 Electric current is allowed to flow through the gel a DNA pieces travel because they are negatively charged and the smallest



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