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Let’s play the review game!

Let’s play the review game!. ( a.k.a how much did you forget this summer?). LIGHTNING REVIEW!! ONs. What’s an ON? Why do we do ONs? (in other words, why is Vershon making us do manual labor when he’s already got the cDNA sequence??). Were you right?.

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Let’s play the review game!

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  1. Let’s play the review game! (a.k.a how much did you forget this summer?)

  2. LIGHTNING REVIEW!! ONs • What’s an ON? • Why do we do ONs? • (in other words, why is Vershon making us do manual labor when he’s already got the cDNA sequence??)

  3. Were you right? • Purpose of an overnight is to amplify cDNA (for use in minipreps) – transforming plasmids with inserts into bacteria is a convenient way to get clones of specific insert • Vershon can’t sequence the cDNA directly because… • He needs more copies • He needs to make sure the cDNA insert is worth sequencing

  4. Million Dollar Question: Which colony should I pick??

  5. “A” is for A+ • We pick white colonies for a reason • Two kinds of plasmids present:

  6. Then what? • Suspend colonies in 2mL of LB Amp (ampicillin to select for transformed bacteria) • Incubate overnight at 37˚C to allow bacteria to proliferate • Vigorous shaking to aerate culture and maintain max surface area

  7. Everybody <3 PCR! • Purpose? • To amplify the insert DNA even further to get a good band in a gel, like so:

  8. 2π is the magic number! • …Actually it’s 360bp. If an insert is <360 bp long, it’s not worth sequencing. • What you need • Nucleoside Triphosphates • Forward and Reverse Primers • Taq Polymerase • The DNA

  9. Lightning Review!! PCR details • Initial Denaturation • 94˚C for 5 minutes Lyse cells, separate DNA strands • Amplification • 94˚C for 30 seconds • 52˚C for 30 seconds (anneal primers) • 72˚C for 1 minute (nucleotides added) • Rinse, Lather and Repeat … 30 times • Additional Elongation • 72˚C for 5 minutes Ensures all DNA strands are full length

  10. Gel Electrophoresis: - • Pretty straightforward +

  11. Priming you on Primers

  12. Determining Insert Size – pop quiz!

  13. Minipreps • Purpose • To purify the DNA for sequencing

  14. Lysis buffer, NaOH; denatures all double strands Schematic Overview Degrades RNA Supernatant = just LB amp Plasmids to renature; net

  15. Restriction Digests • Purpose: To verify the size of the insert • Restriction enzymes = molecular scissors that cut the DNA at specific sites • We use restriction enzyme PvuII; two sites where PvuII can cut: • How many bands would you expect to see in the uncut lane?

  16. Digest this! 10.0 µl of DNA • The two most important rules in enzymes • Always keep enzymes on ice or in a cooler. • Always use a fresh tip when pipetting from the enzyme stocks.

  17. Verifying the Size of the Insert • We know the size of the plasmid vector: • 2900 bp • We know the total bp upstream and downstream the insert after cutting • 700 bp • Rule of thumb – subtract 700 from the smallest band on a gel

  18. Example 2900 bp 1200 bp 400 bp

  19. What happened?

  20. Do they agree?

  21. Questions?

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