Giles 23 April 09

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Giles 23 April 09

  1. 1. Analysis of transcription related H3/H4 displacement at the individual gene level<br />
  2. 2. Following to be done for ArdC, ProP and AltB at mid and late G2<br />Quantification of transcription<br />Quantification of PolII occupancy <br />Quantification of H3/H4 replacement <br />Perform high resolution mapping at mid and late G2 for AltB<br />Experiments<br />
  3. 3. Showed change in AltB expression as expected<br />Showed possible enrichment of PolIIon AltB in late G2<br />Previously...<br />Late G2<br />Mid G2<br />Imp UnIP<br />Imp Un IP<br />
  4. 4. Following to be done for ArdC, ProP and AltB at mid and late G2<br />Quantification of transcription<br />Quantification of PolII occupancy <br />Quantification of H3/H4 replacement <br />Perform high resolution mapping at mid and late G2 for AltB<br />Experiments<br /><br /><br />
  5. 5. Mapping of AltB<br />-230<br />-156<br />
  6. 6. Comparison of mononucleosomes from control and flag incorporated cells<br />Testing effect of Flag histone incorporation<br />
  7. 7. Determine relative amplification from DNA cut 5 min and 15 min compared to uncut<br />Testing Mnase sensitivity of AltB<br />
  8. 8. AltBnucleosomepostition in Mid (off) and Late (on) G2<br />-230<br />-156<br />100bp<br />
  9. 9. Confirmation of quantitative mapping<br />-230<br />-156<br />Primer extension assay<br />
  10. 10. Development of a novel method for high resolution mapping<br />-230<br />-156<br />Tried to quantify/amplify of different products in primer extension reaction<br />Late G2<br />Mid G2<br />PE Q1 Q2 Q3 Q4 Q5<br />PE Q1 Q2 Q3 Q4 Q5<br />
  11. 11. Single primer tests<br />-230<br />-156<br />Neat 1:100<br />Neat 1:100<br />Mid G2<br />Late G2<br />P1/P2 P1 P2<br />P1/P2 P1 P2<br />P1/P2 P1 P2<br />P1/P2 P1 P2<br />
  12. 12. Use reconstituted 5s nucleosomes<br />Confirm position <br />restriction digest<br />New method<br />Apply new method to map completely AltB<br />Validation of new mapping method<br />

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