Let’s play the review game!

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?

22. Questions?