Vacation slides from Bio 437

1. Vacation slides from Bio 437

2. Experiments • 1 Plasmid matching • 2 Amplification & sequencing • 3 Colorful mutations • 4 Designer resistance • 5 Southern bands • 6 Clever cloning

3. Plasmid matching • Given 4 unlabeled plasmid samples and 4 plasmid maps match maps to samples • Use gel separation of restriction fragments to match samples to maps

4. pRCD69

5. pGMC200-ACT1p(rev)

6. pProb-hydrolaseAGS1

7. pProb-synthaseAGS1

8. Restriction enzymes • Had to decide which restriction enzymes to use before starting lab • Could use single or double digest • Wanted restriction fragments such that when ran on gel patterns were unique/identifiable • Make a list of number of times each enzyme cuts each plasmid

9. Table

10. Simplified restriction maps

11. Predicted gel

12. Actual gel

13. Amplification & Sequencing • Amplify and sequence a 300 bp section of a Twinscan predicted C. neoformans gene • Design primers to amplify specified region (by hand!) • Add SpeI site to ends of PCR primers so product can be ligated into RNAi vector later

14. cDNA preperation • Start with C. neoformans RNA sample prepared by Amy • Treat with DNase I to remove genomic DNA • Synthesize cDNA from RNA using Oligo dT primers and RT • Degrade RNA strands with Rnase H

15. PCR • 25 cycles of PCR to amplify sequence from cDNA prepared in last step • Actually did 10 PCR reactions varying amount of primer, Mg concentration, leaving out PCR buffer etc…

16. Reaction table

17. PCR gel check

18. TOPO cloning • Insert PCR product into vector • PCR with Taq leaves 3’ overhanging As • Can insert product into a vector with overhanging 3’ Ts • Induce E. coli to take up plasmids • Good way to store/manipulate PCR products

19. TOPO vectors

20. DNA Miniprep • After growing TOPO containing E. coli extract plasmids • Lyse cells and precipitate out proteins with Potassium acetate/acetic acid • Linear DNA gets tangled in precipitating protein mixture and goes out too • Circular plasmid DNA only thing left in solution

21. Miniprep procedure

22. Sequencing Insert • Sequence off of universal primers in TOPO vector • Already know how this works! • Sequenced with an old slab gel sequencer in the bio department (not GSC) • Also did runs with 2 templates and both primers

23. Resulting alignment

24. Site Directed Mutagenesis • Start with DNA in non-methylated plasmid • Design PCR primer nearly complimentary to mutation site (change desired bases here) • Extend primer in PCR like reaction to copy rest of plasmid without methylation • Digest methylated DNA • Seal nick at edge of mutagenic primer

25. Mutagenesis

26. Base changes

27. Transfomration • Transform plasmids into E. coli cells • Plate onto antibiotic plates to select for uptake of plasmid • Do both mutated and non-mutated plasmids • Non-mutated  red • Mutated  orenge

28. Results • Total failure! • LB broth used to grow bacteria in our room became contaminated with antibiotic resistant bacteria which was more robust than the transformed bacteria • Lawn of bacteria on plates which were neither red nor orange and stunk up lab for days

29. Designer Resistance • Cut 2 plasmids to generate compatible sticky ends and different antibiotic resistances • Let them ligate together randomly and try to get a plasmid that is resistant to both antibiotics

30. Plasmids

31. Simp Plamids

32. Digression • Were first asked to enumerate all potential ligation products • The all seemed like a problem to me

33. Digression • Complete restriction digest gives you 4 linear DNA fragments (8 if you count reverse compliments) • Partial digestion throws in 4 more • Fragments with compatible sticky ends can ligate together

34. Complete

35. Partial

36. Classes of Fragments • This defines 4 classes of fragments •   H ---------B {a, b’, c, d’} •   H----------H {e, g} •   B----------B {f, h} • ’  B----------H {a’, b, c’, d}

37. Building Fragments • All ligation products must be made up of these building blocks • Ligation products can be defined as strings of fragments (like ab or fh) • Rules for building ligation products actually derive from interaction between classes of fragments

38. Solution • This is really a context free grammar problem! • Derivation rules •    |  • ’  ’ |  •   ’ |  •   ’ | 

39. Example • All circularizable plasmids are either  or  • A derivation corresponds to several ligation products

40. Example •  • ’ • ’ • ’ • c f b g • H---BB---BB---HH---H

41. Derivation

42. Rest of experiment • Didn’t get any bacteria that were doubly resistant • Same story as in 3, other rooms reactions worked ours didn’t • First question was the highlight of the project!