(STILL) LOST IN TRANSFECTION Matthew Downs || 4/5/10.

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(STILL) LOST IN TRANSFECTION Matthew Downs || 4/5/10

Transfection Resource  ransfxn.pdf ransfxn.pdf

Linear vs Supercoiled  What the Literature States:  Linear -> Stable  Supercoiled -> Transient  What Professionals say:  It doesn’t matter (honestly it doesn’t)

Dueling Promoters  Double edged sword  Leaving endogenous promoter in reduces experimental time but increases chance of translation not working correctly  Location of ATG can create issues  If ATG comes after the first promoter, but before the endogenous promoter there will be issues  If ATG comes after both the first promoter and the endogenous promoter, there will be no issues with expression.

Current Issues  Jen’s Transfection: Multiple Promoters  Most likely there is ATG between both promoters  Eugene’s Transfection: Localization  SSTR3 Most likely does not localize

Future Planning  1: Research Localization  If you’re trying to localize a maker to a specific part of the cell, make sure your cell has that localization  2: Clean Vectors  Have only what you need in a vector (either with linear or supercoiled)  Reduce the amount of ATG to only the areas where you need it to copy a region

Future Planning  3: Promoters  One is safer  Do you need a strong promoter?  4: Reagents / Optimization  Lipofectamine 2000 (suggested)  Time, Ammount of DNA, Charge ratio