Targefect-BAC  ( Download PDF )

Upper Panel:  Transfection of 170 Kb BAC expression vector expressing green fluorescent protein into Vero cells using the Targefect-BAC kit (combination of Targefect-BAC and virofect).

 

Lower panel: Transfection of HEK-293 cells with BAC DNA using the Targefect BAC reagent (without Virofect)

Data courtesy of Dr. Fuchen Zhou and Dr S. Gao, University of Texas Health Science Center at San Antonio, TX

The Targefect-BAC transfection kit is a combination of the Targefect-BAC) and the Virofect for optimal transfection of BAC-DNA vectors (greater than 150 Kb).

Set up cells to be transfected so that they are about 70% confluent at the time of the experiment.  Store Virofect at -20oC or -70 o C.  Store the Targefect-BAC reagent at 4oC. Do not vortex it and freeze it.

Prepare transfection complexes as follows:
Use clear plastic tubes for complex formation. 

Complexing conditions:
USE HIGH GLUCOSE DMEM (Dulbecco’s modified eagle’s medium containing 4500 mg/liter glucose).  Make additions as follows

Tube #

DMEM (high glucose)

DNA

Targefect-BAC

 

Virofect

1

0.5 ml

10 ug

25 ul

-

2

0.5 ml

6 ug

12 ul

25 ul

3

0.5 ml

4 ug

12 ul

25 ul

4

0.5 ml

10 ug

25 ul

25 ul

Add DMEM first, then add DNA, mix well by flicking the tube about 12 times to create a vortexing action.  Add Targefect-BAC next, mix well again by flicking the tube.  Add Virofect last and mix again. Incubate the tubes at 37 o C for 25 minutes to form the transfection complexes. 

Add 0.25 ml of the transfection mix to 2 ml of complete media for 1 well for a 6-well dish (or for a 35 mm dish).  Prepare 0.5 ml of transfection complex per 60 mm dish and 1 ml of transfection complex per 100mm dish.  Swirl the dish to mix transfection complexes with the cell culture media (with 5-10% serum).  Incubate overnight at 37 o C in CO2 incubator.  Replace the media with fresh complete media the next morning and assay at 36-48 hrs post-transfection. 

 

VIROFECT ENHANCER:

Once the optimal ratio of DNA to Targefect-BAC has been established repeat that experiment titrating the amounts of Virofect enhancer in the transfection complex (enhancer to be added just after the targefect-BAC reagent).

Expected efficiency –30-40%, cells tested, Vero cells, 60% in HEK-293 cells

Please do not use the Virofect enhancer when transfecting HEK-293 cells.  For transfecting BAC DNA into HEK-293 cells use the Targefect-BAC reagent alone or the Targefect-293 reagent

This kit (catalog #BAC-01) contains sufficient reagent for performing 200 transfections in 12-well dishes or 100 transfections in 6-well dishes

Product No.

Quantity

TS list Price

BAC-01

1

$250

BAC-10

10

$2000

Contents:  2 solutions
        – Targefect-BAC and Virofect

Cell lines tested:  Vero, HEK-293, Hela and HUVECs