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Spec statements: Fragments of DNA can be produced by

Spec statements: Fragments of DNA can be produced by Conversion of mRNA to cDNA , using reverse transcriptase. Cutting DNA at specific, palindromic recognition sequences using restriction endonucleases. The polymerase chain reaction (PCR)

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Spec statements: Fragments of DNA can be produced by

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  1. Spec statements: Fragments of DNA can be produced by • Conversion of mRNA to cDNA, using reverse transcriptase. • Cutting DNA at specific, palindromic recognition sequences using restriction endonucleases. • The polymerase chain reaction (PCR) Fragments of DNA produced by the above methods can be used to clone genes by in vivo and in vitro techniques.

  2. Gene Cloning Lesson 2 Polymerase Chain Reaction

  3. Todays lesson-Making this gene into a useable form.

  4. Outcomes -Describe the purpose and principals behind gel electrophoresis -Order the steps in Polymerase Chain Reaction -Construct a flow chart to show how to amplify a gene produced by a restriction endonuclease.

  5. Gel Electrophoresis • Technique that allows us to identify the size of DNA fragments. • We can compare this with a known sample of the gene to check that the fragments are the correct size (the gene that we have isolated is the correct gene).

  6. Gel Electrophoresis • http://learn.genetics.utah.edu/content/labs/gel/ • DNA is negatively charged so it will move to the positive electrode (-ive charge comes from the phosphate group) • Smaller fragments move further along the gel as there is less resistance. • Each band will contain many fragments of DNA that are of the same size. • To see the DNA you need to stain the gel. You could do this with a dye after you have run the gel or use a radioactive marker that you attach to the DNA before hand.

  7. Southern Blotting. • Process that allows the required DNA to be extracted from the electrophoresis gel. • The electrophoresis gel is pressed onto a membrane. • The DNA is passed onto the membrane and it can be easily extracted from here.

  8. Polymerase Chain Reaction. • Now that you have the DNA, you want to make as many copies as possible for insertion into plasmids etc. Polymerase chain reaction does this! Task 1. Order chopped up statements on PCR. Task 2. Check order using page 239-240 of text book. Task 3. Complete PCR summary sheet.

  9. Summary • Create a flow chart that starts with “fragments of DNA made by restriction endonucleases” and ends with “many copies of the required gene”.

  10. Fragments of DNA made by REs. • Electrophoresis of the DNA. The DNA is matched with a known copy of the required gene. • Southern blotting. The DNA is removed from the electrophoresis gel onto a membrane from which it can be easily lifted. • PCR. • Many copies of the required gene are produced.

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