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Which sizes of DNA fragments do you expect to get from the digestion?

Journal 12.09.10:. Which sizes of DNA fragments do you expect to get from the digestion? 2. How can one use the electrophoresis procedure to estimate the size of the DNA fragments?. Before the journal… Add 2 ul of “Loading dye” into your A- and A+ samples.

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Which sizes of DNA fragments do you expect to get from the digestion?

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  1. Journal 12.09.10: • Which sizes of DNA fragments do you expect to get from the digestion? • 2. How can one use the electrophoresis procedure to estimate the size of the DNA fragments?

  2. Before the journal… • Add 2 ul of “Loading dye” into your A- and A+ samples. • Load the entire samples (12ul or less) onto the assigned gels: • Markers (5-10ul), A-, A+. • Cautions: Gel direction / wells are closed with no bubbles!

  3. EcoRI HinDIII BamH1 2300 4000 3000 5000 bp • Which fragment sizes will result from cutting with: • A. HinDIII alone? B. EcoE1 alone? • C. Both HunDIII+EcoR1 • 2. Draw the sizes into the gel.

  4. E H B 2300 4000 3000 5000 bp E+H E H B 23000 bp EcoRI 9000 bp 6000 bp HinDIII 4000 bp 2300bp BamH1 2000 bp 500 bp

  5. http://www.teachersdomain.org/asset/biot11_vid_isolatedna/

  6. No journal today… Per group: Answer questions in page 4a.4 In parallel – observe the gel on the trans-illuminator..

  7. pARA-R construct Recombinant plasmid of interest pARA-R 4720 bp pARA-R 4720 bp BamH I rfp 702bp rfp 702bp Hind III

  8. Gel Electrophoresis - Detection We run the gel until the yellow dye runs out. Wells

  9. After running the gel (till the yellow dye runs out), we stain it in Ethidium bromide, which will fluoresce when bound to DNA:

  10. Supercoiling Multimers “nicked-circle” circle

  11. Structural forms of plasmid Slower than nicked. “multimer” “nicked-circle” Linear (Digested) Supercoiled

  12. A- A+ 10 Kb Ladder 10 Kb Ladder 10 Kb Ladder Multimer Nicked Super Coiled 5 Kb Linear Fragment Linear Fragment

  13. M A- A+ Yellow highlighter shows group number.. Expected results

  14. Lab 2a (restriction) and 4a (electrophoresis) almost always come together – why is that? • 2) Point out at least one thing that you learned so far that is important to know when working in a biotech lab.

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