Haynes Lab:Notebook/Short Projects/2014/10/02

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10/02/14 - Meli'sa Crawford

  • New approach for analyzing sequences


New approach for Analyzing DNA sequences

  • Instead of using the conventional approach for analyzing DNA sequences through ApE, we aligned our plasmids in Benchiling and searched for missing construct parts. We have an idea of the parts that should be in each plasmid:


BD001: 5xGal4-spacer-HSVtkTATA-K-CFP-CFP-NLS-Stop-PolyA

BD002: same as BD001 but inserted into vector V0200.

BD003: 5xGal4-spacer-CMV-Kozak-AmCyan-AmCyan-NLS-Stop-PolyA

BD004: 5xGal4-spacer-HPK-Kozak-AmCyan-AmCyan-NLS-Stop-PolyA

BD005: 5XGAL4-spacer-CMV-Kozak-AmCyan-AmCyan-NLS-Stop-PolyA

BD006: DPRE-5xGal4-spacer-CMV-Kozak-AmCyan-AmCyan-NLS-Stop-PolyA



Plasmids BD003-BD006 were checked through Benchiling. The results are the following:

DNA Missing Parts
BD003 5xGal4
BD003-2 PolyA
BD004 HPK
BD004-2 PolyA
BD005 N/A
BD005-2 PolyA
BD006 5xGal4, Spacer, CMV, Kozak, AmCyan
BD006-2 PolyA

Conclusion

  • BD006- It seems to have D11.12 region only. 5xGal4, Spacer, CMV, Kozak and AmCyan seem to be missing.
  • Will verify results with Dr. Haynes.

Quick Update

October 4, 2014:

  • PolyA2 is the actual polyA used in the constructs. Realigned the sequences today in Benchiling with RFC23 prefix for the forward sequences and the new polyA and RFC23 suffix for the reverse sequences.
  • DPRE may be longer than 800bp, therefore we will not conclude that BD006 does not have any other parts.


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