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Separating and visualizing DNA

Last class. Separating and visualizing DNA. Experimental design. Type II REs. RE1. GCATGC CGTACG. RE1. GC CGTA. ATGC CG. The RE conundrum. Why doesn’t RE cut up genomic DNA in bacteria? - No nuclear membrane to protect DNA - Genomic DNA has multiple RE sites. ?

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Separating and visualizing DNA

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  1. Last class Separating and visualizing DNA Experimental design

  2. Type II REs RE1 GCATGCCGTACG RE1 GCCGTA ATGCCG

  3. The RE conundrum Why doesn’t RE cut up genomic DNA in bacteria? - No nuclear membrane to protect DNA - Genomic DNA has multiple RE sites ? - Then no longer protective  ? - Phage DNA not modified, will get degraded

  4. Solving the RE conundrum _____________ the DNA at RE site! Make ___________ at specific times (Why?) gDNA = Protected viral DNA = Degraded! NOTE: ______________ on “C” or “A” residue

  5. The RE conundrum – the solution RE1 RE1 X GCATGCCGTACG GCATGCCGTACG CH3 CH3 RE1 X RE1 CH3 CH3 GCCGTA ATGCCG

  6. Lab 4 Methylase specificity.

  7. Analytical uses of RE DNA 1 = 10000 bp DNA 2 = 10000 bp You have: Pure DNA 1 Pure DNA 2 Unknown DNA: Maybe pure 1/2 or mixture Sequencer is broken! Need a quick answer: What is unknown?

  8. RE Mapping can quickly test sequences 4 Kb RE 1 RE 1 10 Kb 10 Kb RE 1 6 Kb 6 Kb 10 Kb 4 Kb

  9. RE Map Locations of RE sites on DNA

  10. Size + Pattern is important! Known: Circular DNA, Uncut ~ 12Kb Known: RE1 = 6Kb DNA Known: RE2 = 6Kb DNA Known: RE1 + RE2 = 3Kb DNA RE MAP?

  11. RE Mapping example Known: Circular DNA, Uncut ~ 12Kb Known: RE1 or RE3 = 12Kb DNA Known: RE1 + RE3 = 4Kb + 8Kb DNA Known: RE2 = 2Kb + 8Kb DNA Known: RE2 + RE3 = 2Kb + 6Kb DNA Known: RE2 + RE1 = 2Kb + 6Kb DNA RE MAP?

  12. RE Mapping example 1. Size of DNA = _____ KbBecause? RE ___ 2. Map RE1 & RE3 _____ Kb RE ___ RE ___ 3. Map RE2 in relation to RE3 (Total size!) RE ___ 4. Check RE2 + RE1 (total size!) RE ___

  13. RE Mapping example Known: Circular DNA, Uncut ~ 12Kb Known: RE1 or RE3 = 12Kb DNA Known: RE1 + RE3 = 4Kb + 8Kb DNA Known: RE2 = 2Kb + 8Kb DNA Known: RE2 + RE3 = 2Kb + 6Kb DNA Known: RE2 + RE1 = 2Kb + 6Kb DNA RE MAP?

  14. Look for more examples! Getting uncomfortable & learning to teach yourself…. Search “plasmid re mapping examples”! What other search terms might you try?

  15. Understanding Paper 1 What is a miRNA? Why are miRNAs important? Till this paper, how many miRNAs were known?

  16. miRNA characteristics How were new miRNAs identified? Were cDNA & informatics search sufficient to confirm?

  17. Figure 1 A-C What is the purpose?

  18. Figure 1 A-C What is the experiment?

  19. What is a “Northern” blot? http://www.youtube.com/watch?v=KfHZFyADnNg

  20. Figure 1 A-C What is U6? Why is it included?

  21. Figure 1 A-C What are L1, L2, L3, L4, M? Why are they included?

  22. Figure 1 A-C Conclusion/s? Unanswered questions? Objections?

  23. Figure 1 D What is the main purpose?

  24. Figure 1 D Conclusion/s? Unanswered questions? Objections?

  25. Figure 1 E What is the main purpose?

  26. Figure 1 E Conclusion/s? Unanswered questions? Objections?

  27. Table 1 What is summarized?

  28. Figure 2 What is shown? Unanswered questions? Objections?

  29. Paper 1 Conclusion/s? Future experiment/s?

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