Download Mechanical Properties and Response of Microtubules in Primary Cilia

(STILL)
LOST
IN
TRANSFECTION
Matthew Downs || 4/5/10
Transfection Resource

http://www.promega.com/guides/transfxn_guide/t
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